Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

Impediment of Cerebrospinal Fluid Drainage Through Glymphatic System in Glioma

BackgroundCerebrospinal fluid (CSF) plays an important role in maintaining tissue homeostasis in the central nervous system. In 2012, the new CSF outflow pathway, “the glymphatic system,” was discovered. The glymphatic system mediates CSF and interstitial fluid exchange through the perivascular pathway, which eliminates harmful solutes in the brain parenchyma. In recent studies, the importance of the glymphatic system has been demonstrated in healthy and neurodegenerative disease brains. However, there is limited research on the function of the CSF in brain tumors. Intracranial hypertension caused by glioma can affect CSF drainage, which impacts the delivery of chemotherapy drugs via intrathecal injection. This study focused on changes in the glymphatic system and the role of aquaporin 4 (AQP4) in glymphatic transport in glioma.MethodsIn glioma-bearing rats, the effect of tracer infusion on the intracranial pressure (ICP) was evaluated using an ICP microsensor. In vivo magnetic resonance imaging and ex vivo bright field were used to monitor CSF tracer distribution after cisterna magna injection. AQP4 expression was quantitatively detected, and AQP4 in the astrocytes around the vessels was observed using immunofluorescence.ResultsThe ICP of the tumor group was higher than that of the control group and the infusion rate of 2 µl/min did not affect ICP. In vivo and ex vivo imaging showed that the circulation of CSF tracers was significantly impaired in the tumor. High-power confocal microscopy revealed that, in the tumor, the surrounding of AQP4 by Evans Blue was decreased. In both tumor and contralateral areas, data indicated that the number of cluster designation 34 (CD34+) alpha-smooth muscle actin (α-SMA−) veins were more than that of CD34+α-SMA+ arteries. Moreover, in the tumor area, AQP4 in the astrocytes around the vessels was decreased.ConclusionsThese findings indicate that the para-arterial influx of subarachnoid CSF is limited in glioma, especially in those with reduced levels of the fundamental protein AQP4. Our results provide evidence toward a potential new treatment method for glioma in the future.

High-Resolution Micro-Continuum Approach to Model Matrix-Fracture Interaction and Fluid Leakage

Understanding the fundamental mechanism of fracture-matrix fluid exchange is crucial for the modeling of fractured reservoirs. Traditionally, high-resolution simulations for flow in fractures often neglect the matrix-fracture leakage influence on the fracture hydraulic properties, i.e., assuming impermeable fracture walls. This work introduces a micro-continuum approach to capture the matrix-fracture leakage interaction and its effect on the rock fractures’ hydraulic properties. Because of the multiscale nature of fractured media, full physics Navier-Stokes (NS) representation everywhere in the whole domain is not feasible. We thus employ NS equations to describe the flow in the fracture, and Darcy’s law to model the flow in the surrounding porous rocks. Such hybrid modeling is achieved using the extended Darcy-Brinkman-Stokes (DBS) equation. With this approach, a unified conservation equation for flow in both media is applied by choosing appropriate parameters (e.g., porosity and permeability) for the corresponding domains. We apply an accurate Mixed Finite Element approach to solve the extended DBS equation. Various sensitivity analyses are conducted to explore the leakage effects on the fracture hydraulic properties by varying surrounding matrix permeability, fracture roughness, and Reynolds number (Re). Streamline profiles show the presence of back-flow phenomena, where in-flow and out-flow are possible between the matrix and the fractures. Further, zones of stagnant (eddy) flow are observed around locations with large asperities of sharp corners under high Re conditions. Numerical results show the significant effects of roughness and inertia on flow predictions in fractures for both impermeable and leaky wall cases. Besides, the side-leakage effect can create non-uniform flow behavior within the fracture that may differ significantly from the case with impermeable wall conditions. And this matrix-fracture leakage influence on hydraulic properties of rock fractures matters especially for cases with high matrix permeability, high fracture roughness, and low Re values. In summary, we present a high-resolution micro-continuum approach to explore the flow exchange behavior between the fracture and rock matrix, and further investigate the static and dynamic effects, including variable Reynold numbers, mimicking flow near and away from the wellbore. The approach and results provide significant insights into the flow of fluids through fractures within permeable rocks and can be readily applied in field-scale reservoir simulations.

Cerebral Microcirculation, Perivascular Unit, and Glymphatic System: Role of Aquaporin-4 as the Gatekeeper for Water Homeostasis

In the past, water homeostasis of the brain was understood as a certain quantitative equilibrium of water content between intravascular, interstitial, and intracellular spaces governed mostly by hydrostatic effects i.e., strictly by physical laws. The recent achievements in molecular bioscience have led to substantial changes in this regard. Some new concepts elaborate the idea that all compartments involved in cerebral fluid homeostasis create a functional continuum with an active and precise regulation of fluid exchange between them rather than only serving as separate fluid receptacles with mere passive diffusion mechanisms, based on hydrostatic pressure. According to these concepts, aquaporin-4 (AQP4) plays the central role in cerebral fluid homeostasis, acting as a water channel protein. The AQP4 not only enables water permeability through the blood-brain barrier but also regulates water exchange between perivascular spaces and the rest of the glymphatic system, described as pan-cerebral fluid pathway interlacing macroscopic cerebrospinal fluid (CSF) spaces with the interstitial fluid of brain tissue. With regards to this, AQP4 makes water shift strongly dependent on active processes including changes in cerebral microcirculation and autoregulation of brain vessels capacity. In this paper, the role of the AQP4 as the gatekeeper, regulating the water exchange between intracellular space, glymphatic system (including the so-called neurovascular units), and intravascular compartment is reviewed. In addition, the new concepts of brain edema as a misbalance in water homeostasis are critically appraised based on the newly described role of AQP4 for fluid permeation. Finally, the relevance of these hypotheses for clinical conditions (including brain trauma and stroke) and for both new and old therapy concepts are analyzed.

A nonlinear multi-scale model for blood circulation in a realistic vascular system

In the last decade, numerical models have become an increasingly important tool in biological and medical science. Numerical simulations contribute to a deeper understanding of physiology and are a powerful tool for better diagnostics and treatment. In this paper, a nonlinear multi-scale model framework is developed for blood flow distribution in the full vascular system of an organ. We couple a quasi one-dimensional vascular graph model to represent blood flow in larger vessels and a porous media model to describe flow in smaller vessels and capillary bed. The vascular model is based on Poiseuille’s Law, with pressure correction by elasticity and pressure drop estimation at vessels' junctions. The porous capillary bed is modelled as a two-compartment domain (artery and venous) using Darcy’s Law. The fluid exchange between the artery and venous capillary bed compartments is defined as blood perfusion. The numerical experiments show that the proposed model for blood circulation: (i) is closely dependent on the structure and parameters of both the larger vessels and of the capillary bed, and (ii) provides a realistic blood circulation in the organ. The advantage of the proposed model is that it is complex enough to reliably capture the main underlying physiological function, yet highly flexible as it offers the possibility of incorporating various local effects. Furthermore, the numerical implementation of the model is straightforward and allows for simulations on a regular desktop computer.

Simultaneous multicolor DNA-PAINT without sequential fluid exchange using spectral demixing

Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. The oligonucleotide-based SMLM approach DNA-PAINT robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. However, multicolor DNA-PAINT has primarily been realized by Exchange-PAINT that requires sequential exchange of the imaging solution and thus leads to extended acquisition times. To alleviate the need for fluid exchange and to speed up the acquisition of current multichannel DNA-PAINT, we here present a novel approach that combines DNA-PAINT with simultaneous multicolor acquisition using spectral demixing (SD). By using newly designed probes and a novel multichannel registration procedure we achieve simultaneous multicolor SD-DNA-PAINT with minimal crosstalk. We demonstrate high localization precision (3 - 6 nm) and multicolor registration of dual and triple-color SD-DNA-PAINT by resolving patterns on DNA origami nanostructures and cellular structures.

Association between the use of exchange devices for peritoneal dialysis fluids and peritonitis incidence: A nationwide cohort study

Background: The use of exchange devices for peritoneal dialysis (PD) fluids is a common practice in Japan. Evidence on the effectiveness of exchange devices in preventing PD-related peritonitis is scarce. We evaluated the association between the use of exchange devices for PD fluids and peritonitis incidence. Methods: We retrospectively enrolled 3845 patients, aged ≥20 years, receiving PD for ≥3 months, with available data on the exchange procedure for PD fluids and peritonitis incidence that was obtained from the Japan Renal Data Registry, a nationwide annual survey. The patients were grouped according to whether the manual or device PD fluid exchange method was used. The onset of peritonitis was defined as a leukocyte count of >100/µL (neutrophils ≥50%) in PD effluents. We applied quasi-Poisson regression analyses to estimate the incidence rate ratio (IRR). Age, sex, PD vintage, body mass index, automated PD use, residual kidney function, comorbidities, haemoglobin and serum albumin were adjusted as potential confounders. Results: Older age, automated PD use, diabetes as comorbidity and lower haemoglobin levels were associated with the use of exchange devices for PD fluids. Patients using devices for PD fluid exchange (69.2%) had an increased risk of peritonitis of 37% (IRR: 1.37, 95% confidence interval (CI): 1.07–1.75) and 28% (IRR: 1.28, 95% CI: 1.00–1.63) in the crude and multivariate adjustment models, respectively. Conclusions: The use of exchange devices for PD fluids and peritonitis incidence showed no favourable association. There may remain possible residual confounding by indication.

Active Cerebrospinal Fluid Exchange System for Treatment of Pyogenic Ventriculitis

ABSTRACT BACKGROUND Despite recent advances in antibiotic treatment, pyogenic ventricular brain infections are still associated with adverse clinical outcome in 80% of affected patients and mortality rates approaching 60%. The limitation of antibiotic penetration into the cerebrospinal fluid (CSF) challenges the treatment. Intrathecal treatment remains an option for adjunctive therapy to intravenous (iv) antibiotics when the iv therapy fails to sterilize the CFS. Current treatment options do not allow for changing the CSF composition without adversely affecting intracranial pressure (ICP) and power of hydrogen (pH). OBJECTIVE To investigate if CSF composition exchange has impact on ventriculitis patients. METHODS We report 2 cases with pyogenic ventriculitis treated with a new intracranial active fluid exchange system that consists of a dual-lumen catheter to facilitate irrigation and drainage coupled with an intelligent digital pump. RESULTS This new technique allowed us to change the composition of CSF to an antibiotic-consisted fluid. This resulted in the ability to directly modify the concentration of the targeted antibiotics in the CSF, while simultaneously removing bacterial mass without harming brain tissue and controlling ICP and pH. CONCLUSION Our reported experience shows that drainage of purulent fluid caused by healthcare-associated ventriculitis or meningitis is now possible without harming brain tissue and ICP while also changing the composition of CSF to an antibiotic-consisted fluid. Actively removing pus and altering CSF in this manner had an impact on infection treatment and antibiotic penetration. Further cases are needed to confirm that our treatment algorithm is correctly tailored to assist clinicians in reliably treating this catastrophic condition.

An Experimental Investigation of Mass Transfer in Tight Dual-Porosity Systems

During gas injection in ultra-tight fractured reservoirs, molecular diffusion can play a dominant role in the mass transfer process and enhance recovery by extracting oil components from matrix and delaying gas breakthrough. There has been a growing interest from scholars and operators to study the effect of diffusive mass transfer on the potential incremental recovery from CO2 and rich gas injection. However, many fundamental questions pertaining to the physics of multicomponent multiphase flow and transport are still left unanswered. This paper aims to improve the understanding of multicomponent diffusive mass transfer between matrix and fracture blocks through experimental and modeling work. Displacement experiments were carried out using analog fluids and mesoporous medium to effectively isolate and study the relevant physical mechanisms at play. The experiments were performed in packed columns utilizing silica-gel particles that have internal porosity. The particle size is 40-70 micron with highly controlled internal pore size of 6 nm that makes up approximately 50% of the overall porosity. The quaternary analog fluids system consists of Water, Methanol, Isopropanol, and Isooctane, was used because it mimics the phase behavior of CO2, Methane, Butane and Dodecane mixtures at 2,280 psi and 100°C. Our selection of the analog fluid system and porous medium allowed us to investigate matrix-fracture fluid exchange as observed during an enhanced recovery operation in an ultra-tight fractured system. The effluents from these displacement experiments served as the basis for our analysis of diffusive mass transfer. The role of molecular diffusion in the displacement experiments was investigated by first performing separate diffusion experiments to obtain diffusion coefficients for all relevant binary mixtures. Infinite dilution diffusion coefficients were measured for all binary mixtures and then used to model binary and multicomponent diffusion coefficients over the whole composition range. The accuracy of this approach was determined by performing additional binary diffusion experiments over a broader range of compositions. The displacement experiments were simulated using an in-house simulator and excellent agreement was obtained: The extensive experimental/modeling work related to the diffusion coefficients of the analog fluid system was used in interpreting the diffusive mass transfer between the matrix (stagnant) and fracture (flowing) domains via a 1D linear model. The presented work provides new insights into the role of diffusive mass transfer in ultra-tight fractured systems and builds a framework to highlight the critical data needed to effectively characterize and simulate recovery from such complex geological settings.

The Colloid Osmotic Pressure Across the Glycocalyx: Role of Interstitial Fluid Sub-Compartments in Trans-Vascular Fluid Exchange in Skeletal Muscle

The primary purpose of these investigations is to integrate our growing knowledge about the endothelial glycocalyx as a permeability and osmotic barrier into models of trans-vascular fluid exchange in whole organs. We describe changes in the colloid osmotic pressure (COP) difference for plasma proteins across the glycocalyx after an increase or decrease in capillary pressure. The composition of the fluid under the glycocalyx changes in step with capillary pressure whereas the composition of the interstitial fluid takes many hours to adjust to a change in vascular pressure. We use models where the fluid under the glycocalyx mixes with sub-compartments of the interstitial fluid (ISF) whose volumes are defined from the ultrastructure of the inter-endothelial cleft and the histology of the tissue surrounding the capillaries. The initial protein composition in the sub-compartments is that during steady state filtration in the presence of a large pore pathway in parallel with the “small pore” glycocalyx pathway. Changes in the composition depend on the volume of the sub-compartment and the balance of convective and diffusive transport into and out of each sub-compartment. In skeletal muscle the simplest model assumes that the fluid under the glycocalyx mixes directly with a tissue sub-compartment with a volume less than 20% of the total skeletal muscle interstitial fluid volume. The model places limits on trans-vascular flows during transient filtration and reabsorption over periods of 30–60 min. The key assumption in this model is compromised when the resistance to diffusion between the base of the glycocalyx and the tissue sub-compartment accounts for more than 1% of the total resistance to diffusion across the endothelial barrier. It is well established that, in the steady state, there can be no reabsorption in tissue such as skeletal muscle. Our approach extends this idea to demonstrate that transient changes in vascular pressure favoring initial reabsorption from the interstitial fluid of skeletal muscle result in much less fluid exchange than is commonly assumed. Our approach should enable critical evaluations of the empirical models of trans-vascular fluid exchange being used in the clinic that do not account for the hydrostatic and COPs across the glycocalyx.