High-resolution Fluorescence Imaging Research Articles

Ln3+-doped nanoparticles with enhanced NIR-II luminescence for lighting up blood vessels in mice.

Probes functioning in the second near-infrared window (1000-1700 nm, NIR-II) exhibit higher resolution and diminished auto-fluorescence compared to those in the traditional NIR region (700-950 nm). Here, we designed and synthesized rare earth ion doped probes with core/shell/shell structures and bright luminescence in the NIR-II region excited at 808 nm. With the doping of Ce3+ ions, the emission intensity of Er3+ at 1530 nm increased 10 times, while the upconversion luminescence decreased to less than 1%. After being modified with polyacrylic acid and polyethylene glycol, the as-obtained water-soluble probe exhibits continuous high-resolution for distinguishing 0.25 mm blood vessels even 10 h after injection. Noteworthily, the imaging of tumors was achieved by injecting the probe, indicating that the designed NIR-II probe has sufficient brightness and the ability to passively target tumor tissue.

DeepSynth: Three-dimensional nuclear segmentation of biological images using neural networks trained with synthetic data

The scale of biological microscopy has increased dramatically over the past ten years, with the development of new modalities supporting collection of high-resolution fluorescence image volumes spanning hundreds of microns if not millimeters. The size and complexity of these volumes is such that quantitative analysis requires automated methods of image processing to identify and characterize individual cells. For many workflows, this process starts with segmentation of nuclei that, due to their ubiquity, ease-of-labeling and relatively simple structure, make them appealing targets for automated detection of individual cells. However, in the context of large, three-dimensional image volumes, nuclei present many challenges to automated segmentation, such that conventional approaches are seldom effective and/or robust. Techniques based upon deep-learning have shown great promise, but enthusiasm for applying these techniques is tempered by the need to generate training data, an arduous task, particularly in three dimensions. Here we present results of a new technique of nuclear segmentation using neural networks trained on synthetic data. Comparisons with results obtained using commonly-used image processing packages demonstrate that DeepSynth provides the superior results associated with deep-learning techniques without the need for manual annotation.

NIR-II Dye-Labeled Cylindrical Polymer Brushes for in Vivo Imaging.

Although many types of second near-infrared (NIR-II) dyes have been developed, the NIR-II dye bearing a single reactive group, which is indispensable for specifically labeling nanomaterials or biofunctional molecules, is still lacking. In this work, a donor-acceptor-donor type NIR-II dye named IR1032 bearing an amino group was synthesized and used to covalently label cylindrical polymer brushes. The labeled polymer brushes (named brushes1032) had densely grafted poly(ethylene glycol) (PEG) chains and exhibited a wormlike morphology. In aqueous medium, brushes1032 had an emission peak at 1032 nm and a quantum yield (QY) of ∼0.13% measured with IR 26 as a reference (QY = 0.05%). We demonstrated that the dense PEG chains in brushes1032 were greatly favorable for their QY by separating the fluorophores and shielding them from the interactions with water. After being injected intravenously into tumor-bearing mice, brushes1032 showed high tumor accumulation and provided high-resolution fluorescence imaging, exhibiting great application potentials in tumor detection.

Human stroma and epithelium co-culture in a microfluidic model of a human prostate gland.

The prostate is a walnut-sized gland that surrounds the urethra of males at the base of the bladder comprising a muscular portion, which controls the release of urine, and a glandular portion, which secretes fluids that nourish and protect sperms. Here, we report the development of a microfluidic-based model of a human prostate gland. The polydimethylsiloxane (PDMS) microfluidic device, consisting of two stacked microchannels separated by a polyester porous membrane, enables long-term in vitro cocultivation of human epithelial and stromal cells. The porous separation membrane provides an anchoring scaffold for long-term culturing of the two cell types on its opposite surfaces allowing paracrine signaling but not cell crossing between the two channels. The microfluidic device is transparent enabling high resolution bright-field and fluorescence imaging. Within this coculture model of a human epithelium/stroma interface, we simulated the functional development of the in vivo human prostate gland. We observed the successful differentiation of basal epithelial cells into luminal secretory cells determined biochemically by immunostaining with known differentiation biomarkers, particularly androgen receptor expression. We also observed morphological changes where glandlike mounds appeared with relatively empty centers reminiscent of prostatic glandular acini structures. This prostate-on-a-chip will facilitate the direct evaluation of paracrine and endocrine cross talk between these two cell types as well as studies associated with normal vs disease-related events such as prostate cancer.

A Dual-modality Smartphone Microendoscope for Quantifying the Physiological and Morphological Properties of Epithelial Tissues

We report a nonconcurrent dual-modality fiber-optic microendoscope (named SmartME) that integrates quantitative diffuse reflectance spectroscopy (DRS) and high-resolution fluorescence imaging (FLI) into a smartphone platform. The FLI module has a spatial resolution of ~3.5 µm, which allows the determination of the nuclear-cytoplasmic ratio (N/C) of epithelial tissues. The DRS has a spectral resolution of ~2 nm and can measure the total hemoglobin concentration (THC) and scattering properties of epithelial tissues with mean errors of 4.7% and 6.9%, respectively, which are comparable to the errors achieved with a benchtop spectrometer. Our preliminary in vivo studies from a single healthy human subject demonstrate that the SmartME can noninvasively quantify the tissue parameters of normal human oral mucosa tissues, including labial mucosa tissue, gingival tissue, and tongue dorsum tissue. The THCs of the three oral mucosa tissues are significantly different from each other (p ≤ 0.003). The reduced scattering coefficients of the gingival and labial tissues are significantly different from those of the tongue dorsum tissue (p < 0.001) but are not significantly different from each other. The N/Cs for all three tissue types are similar. The SmartME has great potential to be used as a portable, cost-effective, and globally connected tool to quantify the THC and scattering properties of tissues in vivo.

Photoactivatable Fluorogens by Intramolecular C-H Insertion of Perfluoroaryl Azide.

Molecules, capable of fluorescence turn-on by light, are highly sought-after in spatio-temporal labeling, surface patterning, monitoring cellular and molecular events, and high-resolution fluorescence imaging. In this work, we report a fluorescence turn-on system based on photoinitiated intramolecular C-H insertion of azide into the neighboring aromatic ring. The azide-masked fluorogens were efficiently synthesized via a cascade nucleophilic aromatic substitution of perfluoroaryl azides with carbazoles. The scaffold also allows for derivatization with biological ligands, as exemplified with d-mannose in this study. This photoinitiated intramolecular transformation led to high yields, high photo-conversion efficiency, and well-separated wavelengths for photoactivation and fluorescence excitation. The mannose-derivatized structure enabled spatio-temporal activation and showed high contrast and signal amplification. Live cell imaging suggested that the mannose-tagged fluorogen was transported to the lysosomes.

In vivo ultrasound-switchable fluorescence imaging

The conventional fluorescence imaging has limited spatial resolution in centimeter-deep tissue because of the tissue’s high scattering property. Ultrasound-switchable fluorescence (USF) imaging, a new imaging technique, was recently proposed to realize high-resolution fluorescence imaging in centimeter-deep tissue. However, in vivo USF imaging has not been achieved so far because of the lack of stable near-infrared contrast agents in a biological environment and the lack of data about their biodistributions. In this study, for the first time, we achieved in vivo USF imaging successfully in mice with high resolution. USF imaging in porcine heart tissue and mouse breast tumor via local injections were studied and demonstrated. In vivo and ex vivo USF imaging of the mouse spleen via intravenous injections was also successfully achieved. The results showed that the USF contrast agent adopted in this study was very stable in a biological environment, and it was mainly accumulated into the spleen of the mice. By comparing the results of CT imaging and the results of USF imaging, the accuracy of USF imaging was proved.

Newly-Engineered Materials for Bio-Imaging Technology: A Focus on the Hybrid System of Ultrasound and Fluorescence

As an emerging technique, ultrasound-modulated fluorescence (UMF), or ultrasound switchable fluorescence (USF) bioimaging has shown promising features to produce deep-tissue and high-resolution fluorescence imaging for biomedical research and health diagnosis. The success of UMF or USF heavily relies on the design of their contrast agents (CAs). We herein surveyed recent advances in the development of such unique CAs, including configuration, mechanism, stability, sensitivity, and selectivity. Meanwhile, UMF or USF instrumentation has emerged as developmental breakthrough technologies to existing bio-imaging techniques. The best performance of UMF or USF bio-imaging requires an interactive response between CAs and the instrument. In this review, the description of UMF or USF instrumentation are also included for clarification and better understanding. Finally, the UMF and USF's performance in bioimaging is evaluated based on signal-to-noise ratio, resolution, imaging depth and speed, using photoacoustic imaging (PAI) as a standard, a well-developed technique of hybrid bio-imaging. Unlike PAI, UMF or USF is still in its early stage. Although results demonstrated a proof-of-concept landmark being reached, significant efforts are needed to improve the performance of UMF or USF.

Real-Time 3D High-Resolution Microscopy of Human Cells on the International Space Station.

Here we report the successful first operation of FLUMIAS-DEA, a miniaturized high-resolution 3D fluorescence microscope on the International Space Station (ISS) by imaging two scientific samples in a temperature-constant system, one sample with fixed cells and one sample with living human cells. The FLUMIAS-DEA microscope combines features of a high-resolution 3D fluorescence microscope based on structured illumination microscope (SIM) technology with hardware designs to meet the requirements of a space instrument. We successfully demonstrated that the FLUMIAS technology was able to acquire, transmit, and store high-resolution 3D fluorescence images from fixed and living cells, allowing quantitative and dynamic analysis of subcellular structures, e.g., the cytoskeleton. The capability of real-time analysis methods on ISS will dramatically extend our knowledge about the dynamics of cellular reactions and adaptations to the space environment, which is not only an option, but a requirement of evidence-based medical risk assessment, monitoring and countermeasure development for exploration class missions.

Rapid orthopyroxene growth induced by silica assimilation: constraints from sector-zoned orthopyroxene, olivine oxygen isotopes and trace element variations in the Huangshanxi Ni–Cu deposit, Northwest China

The Permian Huangshanxi Ni–Cu-hosted intrusion, located in the southern Central Asian Orogenic Belt in NW China, is dominated by cumulus olivine and orthopyroxene. Various types of compositional zonings in cumulate phases were studied using high-resolution synchrotron X-Ray fluorescence imaging, electron microprobe, laser ablation inductively coupled plasma mass spectrometry and secondary ion mass spectrometry, to shed light on the relationship between the orthopyroxene-rich cumulate and sulfide accumulation. Normal, oscillatory, and sector zonings of Cr in orthopyroxene from both lherzolites and olivine websterites were noted, likely due to the different cut orientation of orthopyroxene crystals composed of sector-zoned cores and reverse-zoned rims. Significant Fe–Mg–Al–Si variations found in the exterior regions of the grains do not correspond to the Cr zoning. However, Cr content is positively correlated with Ti–Al contents in interior regions of orthopyroxene, reflecting coupled charge substitution during the development of sector zoning, and indicating diffusion-modified Fe–Mg–Al–Si variation in the exterior regions. Primary Cr-sector zoning in the core is likely the result of rapid in situ growth in a boundary layer at the cumulus stage, probably induced by Si addition from wall rocks and crystallization under supersaturated conditions. In addition, some olivine shows Mg–Fe–Mn–Ca–Cr variations in the rim but not in the core, and both core and rim of olivine show a uniform O isotope composition, illustrating that they have not been modified by post-cumulus processes. Given the distinct difference in O isotopic signatures between mantle-derived melts and crustal materials, olivine O isotope composition could be used to decipher the relative degree of Si addition at crystallization. The Fo value, O isotopic signature and trace element content in the cores of olivine grains from different depths show systematic variations, dividing the sulfide-bearing cumulate into two intervals; the lower and upper lherzolite zones with harzburgitic rocks at the contact. In the lower zone, the olivine δ18O values increase upward from ~ 6 to ~ 7‰ and the oxygen fugacity decreases from QFM to QFM-2, suggesting increasing contamination by more reduced crustal materials. Over the same interval, the modal proportion of orthopyroxene and modal orthopyroxene/olivine ratio increase with increasing depth with little variation in whole-rock Mg#, clearly illustrating that the non-cotectic orthopyroxene proportions resulted from Si addition from the wall rock. In the upper zone, the decrease in δ18O values, and increase in olivine nucleation density, Fo value and oxygen fugacity suggest there was a pulse of sulfide- and olivine-charged magma into the magma chamber, that mixed with resident magma to form a hybrid bottom layer in contact with the early-crystallized orthopyroxene-rich cumulate pile. The sulfide content decreases up-section in the lower zone, and then increases again towards the top of the cumulate sequence. This, combined with the compositional variations with depth, suggests that sulfides in the Huangshanxi Ni–Cu deposit settled downward during at least two sulfide-loaded magma pulses. The fast-growing orthopyroxene-rich cumulate may have acted as a permeability barrier, preventing sulfide percolation into the lower cumulate formed during the first magma pulse. Overall, sector zoning in orthopyroxene and compositional variations in olivine suggest that Si and reduced material addition during contamination is of great importance in forming magmatic Ni–Cu deposits hosted by olivine- and orthopyroxene-dominated intrusions.

Optical tweezers with IRM, TIRF and Widefield: studying cytoskeletalprocesses with or without the need for fluorescence labeling

Microtubules and actin filaments are dynamic cytoskeletal structures that interact with motor proteins and play a fundamental role in many essential biological processes including cell division, cell migration, and mechanosensing.Single‐molecule analysis played a central role in revealing many aspects of these complex and dynamic interactions. During a typical dynamic single‐molecule experiment, cytoskeletal filaments are imaged for an extensive period of time using fluorescence methods. It can be highly desirable to study these individual protein filaments with high contrast and at high temporal resolution but without the need for fluorescence labeling, to make the assay set‐up less laborious, costly and – in some experiments – prevent inducing phototoxicity.Here we present an experimental arrangement that includes optical tweezers in combination with Interference Reflection Microscopy (IRM) and Total Internal Reflection Fluorescence (TIRF) Microscopy. Interference Reflection Microscopy is a recently introduced imaging method that allows visualizing biological structures in 3D without the need of fluorescence labeling and with sensitivity exceeding DIC microscopy. Total Internal Reflection Fluorescence microscopy provides high resolution fluorescent imaging of specimen near the working surface with high signal to noise ratio resulting in improved single‐molecule surface assays. In addition, we demonstrate the faster 2D imaging capabilities obtained – compared to confocal and STED techniques – by introducing widefield microscopy methods to our system.In this work, we will discuss the experimental design and show an overview of the latest results obtained using this single‐molecule approach.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Vivo High-resolution Ratiometric Fluorescence Imaging of Inflammation Using NIR-II Nanoprobes with 1550 nm Emission

Quantitatively imaging the spatiotemporal distribution of biological events in living organisms is essential to understand fundamental biological processes. Self-calibrating ratiometric fluorescent probes enable accurate and reliable imaging and sensing, but conventional probes using wavelength of 400-900 nm suffer from extremely low resolution for in vivo application due to the disastrous photon scattering and tissue autofluorescence background. Here, we develop a NIR-IIb (1500-1700 nm) emissive nanoprobe for high-resolution ratiometric fluorescence imaging in vivo. The obtained nanoprobe shows fast ratiometric response to hypochlorous acid (HOCl) with a detection limit down to 500 nM, through an absorption competition-induced emission (ACIE) bioimaging system between lanthanide-based downconversion nanoparticles and Cy7.5 fluorophores. Additionally, we demonstrate the superior spatial resolution of 1550 nm to a penetration depth of 3.5 mm in a scattering tissue phantom, which is 7.1-fold and 2.1-fold higher than that of 1064 and 1344 nm, respectively. With this nanoprobe, clear anatomical structures of lymphatic inflammation in ratiometric channel are observed with a precise resolution of ∼477 μm. This study will motivate the further research on the development of NIR-II probes for high-resolution biosensing in vivo.

Actin Cytoskeleton and Focal Adhesions Regulate the Biased Migration of Breast Cancer Cells on Nanoscale Asymmetric Sawteeth.

Physical guidance from the underlying matrix is a key regulator of cancer invasion and metastasis. We explore the effects of surface topography on the migration phenotype of multiple breast cancer cell lines using aligned nanoscale ridges and asymmetric sawtooth structures. Both benign and metastatic breast cancer cells preferentially move parallel to nanoridges, with enhanced speeds compared to flat surfaces. In contrast, asymmetric sawtooth structures unidirectionally bias the movement of breast cancer cells in a cell-type-dependent manner. Quantitative analysis shows that the level of bias in cell migration increases when cells move with higher speeds or with higher directional persistence. Live-cell imaging studies further reveal that actin polymerization waves are unidirectionally guided by the sawteeth in the same direction as the cell motion. High-resolution fluorescence imaging and scanning electron microscopy studies reveal that two breast cancer cell lines with opposite migrational profiles exhibit profoundly different cell cortical plasticity and focal adhesion patterns. These results suggest that the overall migration response of cancer cells to surface topography is directly related to the underlying cytoskeletal architectures and dynamics, which are regulated by both intrinsic and extrinsic factors.

Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Neurological Disease Modelling for Spinocerebellar Ataxia Using Zebrafish.

The cerebellum integrates sensory information and motor actions. Increasing experimental evidence has revealed that these functions as well as the cerebellar cytoarchitecture are highly conserved in zebrafish compared with mammals. However, the potential of zebrafish for modelling human cerebellar diseases remains to be addressed. Spinocerebellar ataxias (SCAs) represent a group of genetically inherited cerebellar diseases leading to motor discoordination that is most often caused by affected cerebellar Purkinje cells (PCs). Towards modelling SCAs in zebrafish we identified a small-sized PC-specific regulatory element that was used to develop coexpression vectors with tunable expression strength. These vectors allow for in vivo imaging of SCA-affected PCs by high-resolution fluorescence imaging. Next, zebrafish with SCA type 13 (SCA13) transgene expression were established, revealing that SCA13-induced cell-autonomous PC degeneration results in eye movement deficits. Thus, SCA13 zebrafish mimic the neuropathology of an SCA-affected brain as well as the involved loss of motor control and hence provide a powerful approach to unravel SCA13-induced cell biological pathogenic and cytotoxic mechanisms.

Super-Resolution Microscopy of Phloem Proteins.

Super-resolution microscopy bridges the gap between light and electron microscopy and gives new opportunities for the study of proteins that contribute to phloem function. The established super-resolution techniques are derived from fluorescence microscopy and depend on fluorescent dyes, proteins tagged with GFP variants or fluorochrome-decorated antibodies. Compared with confocal microscopy they improve the resolution between 2.5 and 10 times and, thus, allow a much more precise (co-) localization of membranes, plasmodesmata, and structural proteins. However, they are limited to thin tissue slices rather than intact plant organs and can only show immobilized or only slowly moving targets. Accordingly, the first super-resolution micrographs of the phloem were recorded from fixed tissue which was sectioned using a vibratome or microtome. As with transmission electron microscopy, preparation of phloem tissue for super-resolution microscopy is challenged by the sudden pressures release when dissecting the functional tissue (see Chapter 2 ).This chapter describes a protocol for investigation of proteins in the plasma membranes of sieve elements and companion cells. It illustrates how high-resolution fluorescence imaging can provide information that could not be obtained with confocal or electron microscopy. Further, a brief introduction outlines the theoretical background of super-resolution techniques suitable for phloem imaging and summarizes the findings of the first available super-resolution studies on the phloem. The protocol focusses on the crucial steps for super-resolution microscopy of immunolocalized phloem proteins, adjusted to the use of three-dimensional structured illumination microscopy (3D-SIM).

SuperCLEM: an accessible correlative light and electron microscopy approach for investigation of neurons and glia in vitro.

ABSTRACTThe rapid evolution of super-resolution light microscopy has narrowed the gap between light and electron microscopy, allowing the imaging of molecules and cellular structures at high resolution within their normal cellular and tissue context. Multimodal imaging approaches such as correlative light electron microscopy (CLEM) combine these techniques to create a tool with unique imaging capacity. However, these approaches are typically reserved for specialists, and their application to the analysis of neural tissue is challenging. Here we present SuperCLEM, a relatively simple approach that combines super-resolution fluorescence light microscopy (FLM), 3D electron microscopy (3D-EM) and rendering into 3D models. We demonstrate our workflow using neuron-glia cultures from which we first acquire high-resolution fluorescent light images of myelinated axons. After resin embedding and re-identification of the region of interest, serially aligned EM sections are acquired and imaged using a serial block face scanning electron microscope (SBF-SEM). The FLM and 3D-EM datasets are then combined to render 3D models of the myelinated axons. Thus, the SuperCLEM imaging pipeline is a useful new tool for researchers pursuing similar questions in neuronal and other complex tissue culture systems.

Distinction and characterisation of rice genotypes tolerant to combined stresses of salinity and partial submergence, proved by a high-resolution chlorophyll fluorescence imaging system.

Chlorophyll a fluorescence (ChlF) parameters measured with fluorescence imaging techniques were used to investigate the combined effect of salt and partial submergence stress to understand photosynthetic performance in rice (Oryza sativa L.). ChlF parameters such as maximal fluorescence (Fm), variable fluorescence (Fv=Fm -F0), the maximal photochemical efficiency of PSII (Fv/Fm) and the quantum yield of nonregulated energy dissipation of PSII (Y(NO)) were able to distinguish genotypes precisely based on their sensitivity to stress. Upon analysis, we found the images of F0 were indistinguishable among the genotypes, irrespective of their tolerance to salt and partial submergence stress. On the contrary, the images of Fm and Fv/Fm showed marked differences between the tolerant and susceptible genotypes in terms of tissue greenness and the appearance of dark spots as stress symptoms. The images of effective PSII quantum yield, the coefficient of nonphotochemical quenching (qN) and the coefficient of photochemical quenching (qP) captured under different PAR were able to distinguish the tolerant and susceptible genotypes, and were also quite effective for differentiating the tolerant and moderately tolerant ones. Similarly, the values of electron transport rate, qN, qP and Y(NO) were also able to distinguish the genotypes based on their sensitivity to stress. Overall, this investigation indicates the suitability of chlorophyll fluorescence imaging technique for precise phenotyping of rice based on their sensitivity to the combined effect of salt and partial submergence.

Marker-Free Tracking for Motion Artifact Compensation and Deformation Measurements in Optical Mapping Videos of Contracting Hearts.

Optical mapping is a high-resolution fluorescence imaging technique, which provides highly detailed visualizations of the electrophysiological wave phenomena, which trigger the beating of the heart. Recent advancements in optical mapping have demonstrated that the technique can now be performed with moving and contracting hearts and that motion and motion artifacts, once a major limitation, can now be overcome by numerically tracking and stabilizing the heart's motion. As a result, the optical measurement of electrical activity can be obtained from the moving heart surface in a co-moving frame of reference and motion artifacts can be reduced substantially. The aim of this study is to assess and validate the performance of a 2D marker-free motion tracking algorithm, which tracks motion and non-rigid deformations in video images. Because the tracking algorithm does not require markers to be attached to the tissue, it is necessary to verify that it accurately tracks the displacements of the cardiac tissue surface, which not only contracts and deforms, but also fluoresces and exhibits spatio-temporal physiology-related intensity changes. We used computer simulations to generate synthetic optical mapping videos, which show the contracting and fluorescing ventricular heart surface. The synthetic data reproduces experimental data as closely as possible and shows electrical waves propagating across the deforming tissue surface, as seen during voltage-sensitive imaging. We then tested the motion tracking and motion-stabilization algorithm on the synthetic as well as on experimental data. The motion tracking and motion-stabilization algorithm decreases motion artifacts approximately by 80% and achieves sub-pixel precision when tracking motion of 1–10 pixels (in a video image with 100 by 100 pixels), effectively inhibiting motion such that little residual motion remains after tracking and motion-stabilization. To demonstrate the performance of the algorithm, we present optical maps with a substantial reduction in motion artifacts showing action potential waves propagating across the moving and strongly deforming ventricular heart surface. The tracking algorithm reliably tracks motion if the tissue surface is illuminated homogeneously and shows sufficient contrast or texture which can be tracked or if the contrast is artificially or numerically enhanced. In this study, we also show how a reduction in dissociation-related motion artifacts can be quantified and linked to tracking precision. Our results can be used to advance optical mapping techniques, enabling them to image contracting hearts, with the ultimate goal of studying the mutual coupling of electrical and mechanical phenomena in healthy and diseased hearts.

31 - Phosphorylation of eNOS at Thr(T)495 induces mitochondrial fission and a hyper-proliferative endothelial cell phenotype in pulmonary arterial hypertension (PAH)

Mitochondrial dysfunction and cell proliferation play an important role in the pathological arterial constriction associated with pulmonary arterial hypertension (PAH). The mechanisms underlying this blood vessel remodeling is unclear. Recent studies have implicated a disruption of mitochondrial network dynamics to favor fission in the attenuation of mitochondrial function in PAH. We found that mitochondrial fission, or fragmentation, enhanced in endothelial cells isolated from IPAH patients. Further, this shift of morphology correlated with an increase of aerobic glycolysis, the Warburg effect. We also identified an increase in mitochondrial generated reactive oxygen species. These changes correlated with an increase in phospho(p)-T495eNOS protein levels Interestingly, high-resolution fluorescent imaging showed an increase in p-T495-eNOS levels on the mitochondria. We also found that the adenoviral-mediated overexpression of a phosphoT495eNOS mimic (T495DeNOS) in normal human pulmonary arterial endothelial cells (HPAEC) was sufficient to induce mitochondrial fission, a glycolytic shift, and stimulate proliferation. Mdivi-1, an inhibitor of the major mitochondrial fission regulator, Drp1 significantly attenuated both mitochondrial fission and cell proliferation. Finally, we found that inhibiting cellular glycolysis using the hexokinase 2 inhibitor, 2-deoxyglucose (2-DG), attenuated the proliferation of EC cultured form IPAH patients and sensitized them to TNF-α mediated apoptosis. Thus, we conclude that the phosphorylation of eNOS at T495 is intimately involved in regulating both mitochondrial morphology and the Warburg effect during PAH and the development of the hyper-proliferative anti-apoptotic EC phenotype associated with the development of PAH.