Dual‐wavelength hybrid optoacoustic‐ultrasound biomicroscopy for functional imaging of large‐scale cerebral vascular networks

A critical link exists between pathological changes of cerebral vasculature and diseases affecting brain function. Microscopic techniques have played an indispensable role in the study of neurovascular anatomy and functions. Yet, investigations are often hindered by suboptimal trade‐offs between the spatiotemporal resolution, field‐of‐view (FOV) and type of contrast offered by the existing optical microscopy techniques. We present a hybrid dual‐wavelength optoacoustic (OA) biomicroscope capable of rapid transcranial visualization of large‐scale cerebral vascular networks. The system offers 3‐dimensional views of the morphology and oxygenation status of the cerebral vasculature with single capillary resolution and a FOV exceeding 6 × 8 mm², thus covering the entire cortical vasculature in mice. The large‐scale OA imaging capacity is complemented by simultaneously acquired pulse‐echo ultrasound (US) biomicroscopy scans of the mouse skull. The new approach holds great potential to provide better insights into cerebrovascular function and facilitate efficient studies into neurological and vascular abnormalities of the brain.      

兔急性肠系膜上静脉血栓形成模型建立及其病理学观察

目的 建立稳定的兔急性肠系膜上静脉血栓形成模型,观察其病理学变化.方法 采用扎闭静脉、损伤血管壁、激活凝血因子等方法建立模型,分期观察病理学改变.结果 成模后可见血管腔内明显血栓形成;肠道粘膜上皮细胞水肿至坏死脱落伴大量炎性细胞浸润;血管内皮细胞从凹陷到局部坏死脱落,直至大面积坏死脱落,内皮下基质暴露.结论 本实验可成功建立兔肠系膜上静脉血栓形成模型,病理学改变符合临床,模型稳定可靠.     

Wavelength‐Modulated Differential Photoacoustic Spectroscopy (WM‐DPAS) for noninvasive early cancer detection and tissue hypoxia monitoring

This study introduces a novel noninvasive differential photoacoustic method, Wavelength Modulated Differential Photoacoustic Spectroscopy (WM‐DPAS), for noninvasive early cancer detection and continuous hypoxia monitoring through ultrasensitive measurements of hemoglobin oxygenation levels (StO₂). Unlike conventional photoacoustic spectroscopy, WM‐DPAS measures simultaneously two signals induced from square‐wave modulated laser beams at two different wavelengths where the absorption difference between maximum deoxy‐ and oxy‐hemoglobin is 680 nm, and minimum (zero) 808 nm (the isosbestic point). The two‐wavelength measurement efficiently suppresses background, greatly enhances the signal to noise ratio and thus enables WM‐DPAS to detect very small changes in total hemoglobin concentration (C_Hb) and oxygenation levels, thereby identifying pre‐malignant tumors before they are anatomically apparent. The non‐invasive nature also makes WM‐DPAS the best candidate for ICU bedside hypoxia monitoring in stroke patients. Sensitivity tunability is another special feature of the technology: WM‐DPAS can be tuned for different applications such as quick cancer screening and accurate StO₂ quantification by selecting a pair of parameters, signal amplitude ratio and phase shift. The WM‐DPAS theory has been validated with sheep blood phantom measurements.

Sensitivity comparison between conventional single‐ended signal and differential signal.     

Polarization‐multiplexed,dual‐beam swept source optical coherence tomography angiography

A polarization‐multiplexed, dual‐beam setup is proposed to expand the field of view (FOV) for a swept source optical coherence tomography angiography (OCTA) system. This method used a Wollaston prism to split sample path light into 2 orthogonal‐polarized beams. This allowed 2 beams to shine on the cornea at an angle separation of ~14°, which led to a separation of ~4.2 mm on the retina. A 3‐mm glass plate was inserted into one of the beam paths to set a constant path length difference between the 2 polarized beams so the interferogram from the 2 beams are coded at different frequency bands. The resulting OCTA images from the 2 beams were coded with a depth separation of ~2 mm. A total of 5 × 5 mm² angiograms from the 2 beams were obtained simultaneously in 4 seconds. The 2 angiograms then were montaged to get a wider FOV of ~5 × 9.2 mm².      

Large‐depth‐of‐field full‐field optical angiography

A large‐depth‐of‐field full‐field optical angiography (LD‐FFOA) method is developed to expand the depth‐of‐field (DOF) using a contrast pyramid fusion algorithm (CPFA). The absorption intensity fluctuation modulation effect is utilized to obtain full‐field optical angiography (FFOA) images at different focus positions. The CPFA is used to process these FFOA images with different focuses. By selecting high‐contrast areas, the CPFA can highlight the characteristics and details of blood vessels to obtain LD‐FFOA images. In the optimal case of the proposed method, the DOF for FFOA is more than tripled using 10 differently focused FFOA images. Both the phantom and animal experimental results show that the LD‐FFOA resolves FFOA defocusing issues induced by surface and thickness inhomogeneities in biological samples. The proposed method can be potentially applied to practical biological experiments.      

Whole‐brain microcirculation detection after ischemic stroke based on swept‐source optical coherence tomography

The occurrence and development of ischemic stroke are closely related to cerebral blood flow. Real‐time monitoring of cerebral perfusion level is very useful for understanding the mechanisms of the disease. A wide field of view (FOV) is conducive to capturing lesions and observing the progression of the disease. In this paper, we attempt to monitor the whole‐brain microcirculation in middle cerebral artery occlusion (MCAO) rats over time using a wide FOV swept‐source OCT (SS‐OCT) system. A constrained image registration algorithm is used to remove motion artifacts that are prone to occur in a wide FOV angiography. During ischemia, cerebral perfusion levels in the left and right hemispheres, as well as in the whole brain were quantified and compared. Changes in the shape and location of blood vessels were also recorded. The results showed that the trend in cerebral perfusion levels of both hemispheres was highly consistent during MCAO, and the position of the blood vessels varied over time. This work will provide new insights of ischemic stroke and is helpful to assess the effectiveness of potential treatment strategies.      

Measurements of auto‐antibodies to α‐synuclein in the serum and cerebral spinal fluids of patients with Parkinson's disease

Biomarkers for α‐synuclein are needed for diagnosis and prognosis in Parkinson's disease (PD ). Endogenous auto‐antibodies to α‐synuclein could serve as biomarkers for underlying synucleinopathy, but previous assessments of auto‐antibodies have shown variability and inconsistent clinical correlations. We hypothesized that auto‐antibodies to α‐synuclein could be diagnostic for PD and explain its clinical heterogeneity. To test this hypothesis, we developed an enzyme‐linked immunosorbent assay for measuring α‐synuclein auto‐antibodies in human samples. We evaluated 69 serum samples (16 healthy controls (HC ) and 53 PD patients) and 145 CSF samples (52 HC and 93 PD patients) from our Institution. Both serum and CSF were available for 24 participants. Males had higher auto‐antibody levels than females in both fluids. CSF auto‐antibody levels were significantly higher in PD patients as compared with HC , whereas serum levels were not significantly different. CSF auto‐antibody levels did not associate with amyloid‐β_1–42, total tau, or phosphorylated tau. CSF auto‐antibody levels correlated with performance on the Montreal Cognitive Assessment, even when controlled for CSF amyloidβ_1–42. CSF hemoglobin levels, as a proxy for contamination of CSF by blood during lumbar puncture, did not influence these observations. Using recombinant α‐synuclein with N‐ and C‐terminal truncations, we found that CSF auto‐antibodies target amino acids 100 through 120 of α‐synuclein. We conclude that endogenous CSF auto‐antibodies are significantly higher in PD patients as compared with HC , suggesting that they could indicate the presence of underlying synucleinopathy. These auto‐antibodies associate with poor cognition, independently of CSF amyloidβ_1–42, and target a select C‐terminal region of α‐synuclein.

Read the Editorial Highlight for this article on page 433 .

     

Portable optical‐resolution photoacoustic microscopy for volumetric imaging of multiscale organisms

Photoacoustic microscopy (PAM) provides a fundamentally new tool for a broad range of studies of biological structures and functions. However, the use of PAM has been largely limited to small vertebrates due to the large size/weight and the inconvenience of the equipment. Here, we describe a portable optical‐resolution photoacoustic microscopy (pORPAM) system for 3‐dimensional (3D) imaging of small‐to‐large rodents and humans with a high spatiotemporal resolution and a large field of view. We show extensive applications of pORPAM to multiscale animals including mice and rabbits. In addition, we image the 3D vascular networks of human lips, and demonstrate the feasibility of pORPAM to observe the recovery process of oral ulcer and cancer‐associated capillary loops in human oral cavities. This technology is promising for broad biomedical studies from fundamental biology to clinical diseases.      

High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2‐fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub‐diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro‐vascular dynamics after laser‐induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological‐imaging capacity of any polygon scanning microscope system.

     

Periphyton‐based transfer functions to assess ecological imbalance and management of a subtropical ombrotrophic peatland

1. To assess the biological status and response of aquatic resources to management actions, managers and decision‐makers require accurate and precise metrics. This is especially true for some parts of the Florida Everglades where multiple stressors (e.g. hydrologic alterations and eutrophication) have resulted in a highly degraded and fragmented ecosystem. Biological assessments are required that directly allow for the evaluation of historical and current status and responses to implantation of large‐scale restoration projects. 2. Utilising periphyton composition and water‐quality data obtained from long‐term (15 years) monitoring programmes, we developed calibrated and verified periphyton‐based numerical models (transfer functions) that could be used to simultaneously assess multiple stressors affecting the Everglades peatland (e.g. salinity, nitrogen and phosphorus). Periphyton is an ideal indicator because responses to stressors are rapid and predictable and possess valued ecological attributes. 3. Weighted averaging partial least squares regression was used to develop models to infer water‐quality concentrations from 456 samples comprising 319 periphyton taxa. Measured versus periphyton‐inferred concentrations were strongly related for log‐transformed salinity ( = 0.81; RMSEP 0.15 mg L^?1) and log‐transformed total phosphorus (TP; = 0.70; RMSEP 0.18 mg L^?1), but weakly related for total Kjeldahl nitrogen (TKN) ( = 0.46; RMSEP 0.12 mg L^?1). Validation results using an independent 455 sample data set were similar (log(salinity) r² = 0.78, log(TP) r² = 0.65 and log(TKN) r² = 0.38). 4. Water Conservation Area 1 (WCA‐1), a large ombrotrophic subtropical peatland impacted by multiple water‐quality stressors that has undergone major changes in water management, was used as a case study. The models were applied to a long‐term periphyton data set to reconstruct water‐quality trends in relation to restoration efforts to reduce nutrient loading to the Everglades. The combination of biologically inferred TP and salinity was used to identify the ecological status of periphyton assemblages. Periphyton assemblages were ecologically imbalanced with respect to salinity and TP. Salinity imbalance varied spatially and temporally, whereas TP was spatially restricted. Imbalances caused by water management were owing to salinity more so than to TP. 5. The transfer functions developed for the Everglades are trait‐based quantitative numerical methods and are ideal because the abundances of species are modelled numerically in relation to a stressor. The resulting inferred value is a numerical representation of the stressor’s effect on biological condition that can be compared against the management of the stressor independent of other factors. The benefits are that biological lags or hysteresis events can easily be identified and environmental conditions can be estimated when measurements are lacking. Reporting biological assessments in terms of well‐defined water‐quality metrics (e.g. numeric criterion) increased the communicative ability of the assessment. The use of multiple metrics to assess ecological imbalance increased the ability to identify probable causes.     

Tracking mesenchymal stromal cells using an ultra‐bright TAT‐functionalized plasmonic‐active nanoplatform

High‐resolution tracking of stem cells remains a challenging task. An ultra‐bright contrast agent with extended intracellular retention is suitable for in vivo high‐resolution tracking of stem cells following the implantation. Here, a plasmonic‐active nanoplatform was developed for tracking mesenchymal stromal cells (MSCs) in mice. The nanoplatform consisted of TAT peptide‐functionalized gold nanostars (TAT‐GNS) that emit ultra‐bright two‐photon photoluminescence capable of tracking MSCs under high‐resolution optical imaging. In vitro experiment showed TAT‐GNS‐labeled MSCs retained a similar differentiability to that of non‐labeled MSCs controls. Due to their star shape, TAT‐GNS exhibited greater intracellular retention than that of commercial Q‐Tracker. In vivo imaging of TAT‐GNS‐labeled MSCs five days following intra‐arterial injections in mice kidneys showed possible MSCs implantation in juxta‐glomerular (JG) regions, but non‐specifically in glomeruli and afferent arterioles as well. With future design to optimize GNS labeling specificity and clearance, plasmonic‐active nanoplatforms may be a useful intracellular tracking tool for stem cell research.

An ultra‐bright intracellular contrast agent is developed using TAT peptide‐functionalized gold nanostars (TAT‐GNS). It poses minimal influence on the stem cell differentiability. It exhibits stronger two‐photon photoluminescence and superior labeling efficiency than commercial Q‐Tracker. Following renal implantation, some TAT‐GNS‐labeled MSCs permeate blood vessels and migrate to the juxta‐glomerular region.     

A simple approach for non‐invasive transcranial optical vascular imaging (nTOVI)

In vivo imaging of cerebral vasculature is highly vital for clinicians and medical researchers alike. For a number of years non‐invasive optical‐based imaging of brain vascular network by using standard fluorescence probes has been considered as impossible. In the current paper controverting this paradigm, we present a robust non‐invasive optical‐based imaging approach that allows visualize major cerebral vessels at the high temporal and spatial resolution. The developed technique is simple to use, utilizes standard fluorescent dyes, inexpensive micro‐imaging and computation procedures. The ability to clearly visualize middle cerebral artery and other major vessels of brain vascular network, as well as the measurements of dynamics of blood flow are presented. The developed imaging approach has a great potential in neuroimaging and can significantly expand the capabilities of preclinical functional studies of brain and notably contribute for analysis of cerebral blood circulation in disorder models.

An example of 1 × 1.5 cm color‐coded image of brain blood vessels of mouse obtained in vivo by transcranial optical vascular imaging (TOVI) approach through the intact cranium.     

Inferring a weighted elastic network from partial unfolding with coarse‐grained simulations

A number of studies have demonstrated that simple elastic network models can reproduce experimental B‐factors, providing insights into the structure–function properties of proteins. Here, we report a study on how to improve an elastic network model and explore its performance by predicting the experimental B‐factors. Elastic network models are built on the experimental coordinates, and they only take the pairs of atoms within a given cutoff distance r_c into account. These models describe the interactions by elastic springs with the same force constant. We have developed a method based on numerical simulations with a simple coarse‐grained force field, to attribute weights to these spring constants. This method considers the time that two atoms remain connected in the network during partial unfolding, establishing a means of measuring the strength of each link. We examined two different coarse‐grained force fields and explored the computation of these weights by unfolding the native structures. Proteins 2014; 82:119–129. © 2013 Wiley Periodicals, Inc.     

Reduction of superior mesenteric hemodynamic responsiveness to [Sar1, Thr8]-angiotensin II and bradykinin, but not sodium nitroprusside, in the presence of homocysteine infusion

Hyperhomocysteinemia (HHcy) has been shown to be an independent risk factor for cardiovascular diseases, superior mesenteric thrombosis and inflammatory bowel disease. Superior mesenteric artery (SMA) supplies the intestine and reduced SMA blood flow results in intestinal ischemia. Although in vitro studies have shown that endothelium-dependent vasorelaxation of SMA is reduced in the presence of homocysteine incubation, it is not confirmed with in vivo studies. In this work, we evaluated responsiveness of SMA to endothelium-dependent or -independent vasodilators and a vasoconstrictor in the absence and presence of acute HHcy in vivo to clarify effect of HHcy on superior mesenteric vascular function. Sodium nitroprusside (SNP), bradykinin (BK), and [Sar1,Thr8]angiotensin II ([Sar1,Thr8]-ANG II) were intravenously administrated in sequence in male Sprague-Dawley rats with or without D,L-homocysteine infusion (6 mg/kg/min) through femoral vein. Agonists-induced changes in carotid artery blood pressure, superior mesenteric blood flow and vascular resistance were measured in the present study. We found that acute HHcy infusion had little effects on SNP-induced hemodynamic changes; however, BK-induced changes in blood pressure, blood flow and vascular resistance were significantly reduced in the presence of HHcy infusion. Additionally, HHcy also markedly decreased [Sar1,Thr8]-ANG II-induced superior mesenteric hemodynamic changes. These results demonstrated that responsiveness of SMA to vasoconstrictor, endothelium-dependent, but not endothelium-independent vasodilator, was inhibited in the presence of Hcy infusion. This HHcy-associated vascular hyporesponsiveness to vasoconstrictors and endothelium-dependent vasodilators may partially contribute to circulatory dysfunctions.     

The biology and polymer physics underlying large‐scale chromosome organization

Chromosome large‐scale organization is a beautiful example of the interplay between physics and biology. DNA molecules are polymers and thus belong to the class of molecules for which physicists have developed models and formulated testable hypotheses to understand their arrangement and dynamic properties in solution, based on the principles of polymer physics. Biologists documented and discovered the biochemical basis for the structure, function and dynamic spatial organization of chromosomes in cells. The underlying principles of chromosome organization have recently been revealed in unprecedented detail using high‐resolution chromosome capture technology that can simultaneously detect chromosome contact sites throughout the genome. These independent lines of investigation have now converged on a model in which DNA loops, generated by the loop extrusion mechanism, are the basic organizational and functional units of the chromosome.      

A Spatial Model of Insulin‐Granule Dynamics in Pancreatic β‐Cells

Insulin secretion from pancreatic β‐cells in response to sudden glucose stimulation is biphasic. Prolonged secretion in vivo requires synthesis, delivery to the plasma membrane (PM) and exocytosis of insulin secretory granules (SGs). Here, we provide the first agent‐based space‐resolved model for SG dynamics in pancreatic β‐cells. Using recent experimental data, we consider a single β‐cell with identical SGs moving on a phenomenologically represented cytoskeleton network. A single exocytotic machinery mediates SG exocytosis on the PM. This novel model reproduces the measured spatial organization of SGs and insulin secretion patterns under different stimulation protocols. It proposes that the insulin potentiation effect and the rising second‐phase secretion are mainly due to the increasing number of docking sites on the PM. Furthermore, it shows that 6 min after glucose stimulation, the ‘newcomer’ SGs are recruited from a region within less than 600 nm from the PM.      

The role of glutamate signaling in incentive salience: second‐by‐second glutamate recordings in awake Sprague‐Dawley rats

The attribution of incentive salience to reward‐predictive stimuli has been shown to be associated with substance abuse‐like behavior such as increased drug taking. Evidence suggests that glutamate neurotransmission and sequential N‐methyl‐D‐aspartate (NMDA) activation are involved in the attribution of incentive salience. Here, we further explore the role of second‐by‐second glutamate neurotransmission in the attribution of incentive salience to reward‐predictive stimuli by measuring sign‐tracking behavior during a Pavlovian conditioned approach procedure using ceramic‐based microelectrode arrays configured for sensitive measures of extracellular glutamate in awake behaving Sprague‐Dawley rats. Specifically, we show that there is an increase in extracellular glutamate levels in the prelimbic cortex (PrL) and the nucleus accumbens core (NAcC) during sign‐tracking behavior to a food‐predictive conditioned stimulus (CS+) compared to the presentation of a non‐predictive conditioned stimulus (CS?). Furthermore, the results indicate greater increases in extracellular glutamate levels in the PrL compared to NAcC in response to the CS+, including differences in glutamate release and signal decay. Taken together, the present research suggests that there is differential glutamate signaling in the NAcC and PrL during sign‐tracking behavior to a food‐predictive CS+.

     

Back Cover: Large‐depth‐of‐field full‐field optical angiography (J. Biophotonics 5/2019)

A large‐depth‐of‐field full‐field optical angiography (LD‐FFOA) method is developed to expand the depth‐of‐field (DOF). The contrast pyramid fusion algorithm is used to fuse 10 FFOA images at different focus depth. Cover images of mouse ear shows LD‐FFOA image has higher contrast and more detailed features. The LD‐FFOA method solves the defocused problem caused by the limited DOF of lens, the curved surface and uneven thickness of the sample. Further details can be found in the article by Mingyi Wang, Nanshou Wu, Hongheng Huang, et al. ( e201800329 ).

     

Effects of distance on detectability of Arctic waterfowl using double‐observer sampling during helicopter surveys

Aerial survey is an important, widely employed approach for estimating free‐ranging wildlife over large or inaccessible study areas. We studied how a distance covariate influenced probability of double‐observer detections for birds counted during a helicopter survey in Canada’s central Arctic. Two observers, one behind the other but visually obscured from each other, counted birds in an incompletely shared field of view to a distance of 200 m. Each observer assigned detections to one of five 40‐m distance bins, guided by semi‐transparent marks on aircraft windows. Detections were recorded with distance bin, taxonomic group, wing‐flapping behavior, and group size. We compared two general model‐based estimation approaches pertinent to sampling wildlife under such situations. One was based on double‐observer methods without distance information, that provide sampling analogous to that required for mark–recapture (MR) estimation of detection probability, , and group abundance, , along a fixed‐width strip transect. The other method incorporated double‐observer MR with a categorical distance covariate (MRD). A priori, we were concerned that estimators from MR models were compromised by heterogeneity in due to un‐modeled distance information; that is, more distant birds are less likely to be detected by both observers, with the predicted effect that would be biased high, and biased low. We found that, despite increased complexity, MRD models (ΔAICc range: 0–16) fit data far better than MR models (ΔAICc range: 204–258). However, contrary to expectation, the more naïve MR estimators of were biased low in all cases, but only by 2%–5% in most cases. We suspect that this apparently anomalous finding was the result of specific limitations to, and trade‐offs in, visibility by observers on the survey platform used. While MR models provided acceptable point estimates of group abundance, their far higher stranded errors (0%–40%) compared to MRD estimates would compromise ability to detect temporal or spatial differences in abundance. Given improved precision of MRD models relative to MR models, and the possibility of bias when using MR methods from other survey platforms, we recommend avian ecologists use MRD protocols and estimation procedures when surveying Arctic bird populations.