Optical Slices Research Articles

Displacement sensing based on microscopic vision with high resolution and large measuring range

AbstractMicroimage strain sensing (MISS) is a novel piston‐type sensor based on microscopic vision. In this study, optical disc slice is used as information carriers to improve MISS. There are multiple pits on the surface of an optical disc. By using machine vision algorithms, the pits can be converted into digital information, making them scales for recording displacements. By this means, we proposed a sensing method that combines high resolution, wide range, and strong robustness. The study measured displacement under different conditions. To address inevitable factors such as pixel drift, and manufacturing errors, corresponding compensation methods were provided. The results show that the measurements closely match those of the linear variable differential transformer, with a resolution of up to 20 nm and a range approaching the sensor size. Despite the sensor's dependence on machine vision, it demonstrates strong resistance to environmental factors such as brightness and angle. Combining compensation methods for pixel drift, and manufacturing errors, this sensor can be well‐applied in various working conditions.

Knockdown of the SDHB Subunit Induces Mitochondrial Remodeling in Human Airway Smooth Muscle Cells

Succinate dehydrogenase (SDH) is a mitochondrial enzyme that serves a dual role in mitochondrial respiration: catalyzing the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle and transferring electrons from succinate to ubiquinone as complex II in the electron transport chain (ETC). SDH is composed of four subunits (SDHA-SDHD) and the loss of SDHB subunit, specifically, has been shown to increase oxidative stress. As a part of the catalytic core of the SDH complex, the SDHB subunit mediates electron transfer to ubiquinone. In human airway smooth muscle (hASM) cells with siRNA-mediated knockdown of SDHB, the maximum velocity of the SDH reaction (SDHmax) is decreased. Moreover, SDHB-knockdown leads to an increase in mitochondrial volume density, indicating a role of SDH in mitochondrial remodeling. However, the impact of SDHB-knockdown on mitochondrial dynamics has not been explored. Therefore, we hypothesize that SDHB-knockdown promotes mitochondrial fragmentation as part of mitochondrial remodeling in hASM cells. To investigate this hypothesis, bronchiolar tissue samples were collected in female and male patients undergoing lung surgery with no current history of smoking or respiratory diseases, the smooth muscle layer was dissected and hASM cells were dissociated. The targeted knockdown of SDHB was achieved by siRNA-mediated knockdown. Mitochondria in hASM cells were labeled with MitoTracker Green (200 nM) and imaged in 3D with a Z optical slice of 0.5 μm using an oil-immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. Optical slices were deconvolved to enhance contrast and the MitoTracker labeling was thresholded to create binary images, which were then reconstructed in 3D. From these images, the mitochondrial complexity index (MCI) was calculated to quantify the extent of fragmentation. To identify the signaling pathway involved in mitochondrial fragmentation, the expression and phosphorylation of dynamin-related protein 1 (DRP1) was quantified by Western blot. Mitochondrial fragmentation is associated with increased phosphorylation of DRP1 at Ser616 (pDRP1s616). Furthermore, mitophagy was assessed by determining the mitochondrial expression of PINK1 and the phosphorylation of Parkin at Ser65 (pParkinS65). Mitochondrial biogenesis was also evaluated through the expression of PGC1α, NRF1/2 and TFAM. Our results show that SDHB-knockdown increased mitochondrial fragmentation (reduced MCI) in hASM cells, and increased pDRP1s616 phosphorylation and its translocation to mitochondria. SDHB-knockdown also increased mitochondrial expression of PINK1 and pParkins65 phosphorylation indicating activation of mitophagy. We also confirmed that SDHB-knockdown increased expression of PGC1α, NRF1/2 and TFAM consistent with mitochondrial biogenesis. These findings unveil the mechanisms underlying SDHB-knockdown-mediated mitochondrial remodeling. Supported by NIH grant R01-HL157984 (GCS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

C2 Spinal Cord Hemisection Reduces Mitochondrial Volume Density in Phrenic Motor Neurons

The diaphragm muscle (DIAm) is the major inspiratory pump for breathing. Neural control of the DIAm during breathing involves the recruitment of fatigue resistant motor units comprising smaller phrenic motor neurons (PhMNs), which innervate type I and IIa fibers. More fatigable DIAm motor units comprise larger PhMNs that innervate type IIx/IIb fibers, which generate greater forces but are more fatigable. The fatigable motor units are only infrequently recruited for expulsive behaviors of the DIAm. Thus, there are marked differences in the activation history of DIAm motor units. Consistent with their higher activity levels, previous studies from our lab have shown that smaller PhMNs and type I and IIa DIAm fibers have greater mitochondrial volume density (MVD). PhMNs located in the cervical spinal cord (C3-C5) receive direct excitatory input from medullary premotor neurons, which is primarily ipsilateral. Thus, C2 spinal cord hemisection (C2SH) disrupts the neural pathway essential for breathing and significantly reduces DIAm activity on the same side. Therefore, we hypothesized that C2SH significantly reduces MVD of smaller PhMNs. In Sprague Dawley rats, PhMNs on both sides were retrogradely labeled by intrapleural injection of cholera toxin B (CTB) 3 days prior to C2SH (right side). In addition, DIAm EMG activity was recorded on both sides by inserting bipolar electrodes. Mitochondria were labeled by intraspinal injection of MitoTracker Red and imaged in identified PhMNs using 3D confocal microscopy. A series of optical slices (0.5 mm) of PhMNs were deconvolved to improve contrast, then thresholded for MitoTracker labeling followed by 3D reconstruction, providing detailed images of mitochondrial morphometry and distribution within PhMNs. In sham rats, the MVD of smaller (lower somal surface area) PhMNs was higher compared to larger PhMNs. C2SH reduced DIAm EMG activity on the ipsilateral side across a 14-day period, while activity on the contralateral side increased. After 14 days, C2SH reduced MVD by ~25% in smaller PhMNs on the ipsilateral side. We conclude that the reduced activity of smaller PhMNs induced by C2SH induced mitochondrial morphological changes, most likely due to reduced mitochondrial biogenesis. These results indicate that reduced DIAm activity associated with mechanical ventilation may impose mitochondrial remodeling that reduces mitochondrial oxidative capacity in PhMNs. These adaptations may disrupt mitochondrial function and impair weaning from mechanical ventilation. Research support by NIH grants HL166204, HL146114 and AG44615. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Marching Windows: Scalable Mesh Generation for Volumetric Data With Multiple Materials.

Volumetric data abounds in medical imaging and other fields. With the improved imaging quality and the increased resolution, volumetric datasets are getting so large that the existing tools have become inadequate for processing and analyzing the data. Here we consider the problem of computing tetrahedral meshes to represent large volumetric datasets with labeled multiple materials, which are often encountered in medical imaging or microscopy optical slice tomography. Such tetrahedral meshes are a more compact and expressive geometric representation so are in demand for efficient visualization and simulation of the data, which are impossible if the original large volumetric data are used directly due to the large memory requirement. Existing methods for meshing volumetric data are not scalable for handling large datasets due to their sheer demand on excessively large run-time memory or failure to produce a tet-mesh that preserves the multi-material structure of the original volumetric data. In this article we propose a novel approach, called Marching Windows, that uses a moving window and a disk-swap strategy to reduce the run-time memory footprint, devise a new scheme that guarantees to preserve the topological structure of the original dataset, and adopt an error-guided optimization technique to improve both geometric approximation error and mesh quality. Extensive experiments show that our method is capable of processing very large volumetric datasets beyond the capability of the existing methods and producing tetrahedral meshes of high quality.

Data-driven decision-making framework for optical fronthaul slice resizing in 6G networks

The third-generation partnership introduced three main types of slices: enhanced mobile broadband, massive machine-type communication, and ultrareliable low-latency communication. To accommodate these services, the next generation of mobile networks will require architecture with distinct requirements and network slices. To implement these services on an optical fronthaul, the slices will be hosted using lightpaths. Such lightpaths will have to accommodate latency and bandwidth constraints to keep radio units (RUs) and baseband units (BBUs) synchronized. However, the traffic in a slice may vary, and the resources allocated to a long-established lightpath could be out of date, leading to waste or lack of resources. For example, a lack of bandwidth can cause desynchronization between BBUs and RUs. Therefore, the slice must be resized regularly to meet the variable demands. This work proposes a data-driven decision-making (DDDM) framework to resize the fronthaul slices while mitigating the consequences of a lack of bandwidth. The framework uses long short-term memory to implement its analytical stage and integer linear programming (ILP) to reconfigure the entire network when it is required. The results show that the DDDM-based framework outperforms the state-of-the-art ILP-based heuristic by up to 15% in terms of radio blocking mitigation.

Features of Peripapillary Hyperreflective Ovoid Mass-Like Structures in Nonarteritic Anterior Ischemic Optic Neuropathy Patients and Normal Controls.

To determine the characteristics of peripapillary hyperreflective ovoid mass-like structures (PHOMS) in patients with nonarteritic anterior ischemic optic neuropathy (NAION) and in normal adults. A total of 406 included eyes were divided into four groups: acute NAION group, chronic NAION group, unaffected group, and normal eyes group. PHOMS were detected on optical coherence tomography slices from optical coherence tomography angiography scans centered on the optic nerve head (ONH). The differences in age, sex, and ONH parameters were investigated between eyes with PHOMS and eyes without PHOMS among groups. The prevalence of PHOMS in acute eyes (43.48%) and fellow eyes (28.20%) was significantly higher than that in normal eyes (11.76%) (acute vs. normal, P < 0.001; fellow vs. normal, P = 0.014). In the acute group, the PHOMS score of size was negatively correlated with age in acute eyes (r = -0.486, P = 0.03). The size of PHOMS was negatively correlated with age and cup/disc ratio and positively correlated with retinal nerve fiber layer thickness in the nasal and inferior sectors in the normal groups. No differences in age, sex, ONH parameters, or visual field defects were found between eyes with PHOMS and eyes without PHOMS. The prevalence of PHOMS increased significantly in acute nonoptic disc drusen (NODD)-NAION eyes and fellow eyes. PHOMS could also be found among normal adults. PHOMS may be a nonspecific sign secondary to ONH edema and axoplasmic stasis. The high prevalence of PHOMS in acute NODD-NAION eyes may indicate axoplasmic stasis secondary to tissue edema.

Acquisition and annotation in high resolution in vivo digital biopsy by confocal microscopy for diagnosis in oral precancer and cancer.

Scanned fibre endomicroscopes are full point-scanning confocal microscopes with submicron lateral resolution with an optical slice thickness thin enough to isolate individual cell layers, allow active positioning of the optical slice in the z-axis and collection of megapixel images. Here we present descriptive findings and a brief atlas of an acquisition and annotation protocol high resolution in vivo capture of oral mucosal pathology including oral squamous cell carcinoma and dysplasia using a fluorescence scanned fibre endomicroscope with 3 topical fluorescent imaging agents: fluorescein, acriflavine and PARPi-FL. Digital biopsy was successfully performed via an acquisition protocol in seventy-one patients presenting for investigation of oral mucosal abnormalities using a miniaturized, handheld scanned fibre endoscope. Multiple imaging agents were utilized and multiple time points sampled. Fifty-nine patients had a matched histopathology correlating in location with imaging. The images were annotated back to macrographic location using a purpose-built software, MouthMap™. Acquisition and annotation of cellular level resolved images was demonstrated with all 3 topical agents. Descriptive observations between clinically or histologically normal oral mucosa showed regular intranuclear distance, a regular nuclear profile and fluorescent homogeneity. This was dependent on the intraoral location and type of epithelium being observed. Key features of malignancy were a loss of intranuclear distance, disordered nuclear clustering and irregular nuclear fluorescence intensity and size. Perinuclear fluorescent granules were seen in the absence of irregular nuclear features in lichenoid inflammation. High resolution oral biopsy allows for painless and rapid capture of multiple mucosal sites, resulting in more data points to increase diagnostic precision. High resolution digital micrographs can be easily compared serially across multiple time points utilizing an annotation software. In the present study we have demonstrated realization of a high-resolution digital biopsy protocol of the oral mucosa for utility in the diagnosis of oral cancer and precancer..

Chemical isotope labeling assisted liquid chromatography-mass spectrometry method for simultaneous analysis of central carbon metabolism intermediates

Central carbon metabolism pathway (CCM) is one of the most important metabolic pathways in all living organisms and play crucial function in aspect of organism life. However, the simultaneous detection of CCM intermediates remains challenging. Here, we developed a chemical isotope labeling combined with LC-MS method for simultaneous determination of CCM intermediates with high coverage and accuracy. By chemical derivatization with 2-(diazo-methyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-DMBA, all CCM intermediates obtain better separation and accurate quantification at a single LC-MS run. The obtained limits of detection of CCM intermediates ranged from 5 to 36 pg/mL. Using this method, we achieved simultaneous and accurate quantification of 22 CCM intermediates in different biological samples. Take account of the high detection sensitivity of the developed method, this method was further applied to the quantification of CCM intermediates at single-cell level. Finally, 21 CCM intermediates were detected in 1000 HEK-293T cells and 9 CCM intermediates were detected in mouse kidney glomeruli optical slice samples (10∼100 cells).

TNF alpha induces mitophagy in human airway smooth muscle cells

Mitophagy is a regulated multistep process of selective removal of dysfunctional/damaged mitochondria by autophagy and is critical for the regulation of mitochondrial quality control in many physiological processes. The best characterized pathway of mitophagy involves PTEN-induced kinase 1 (PINK1) and E3-ubiquitin ligase Parkin. Upon mitochondrial depolarization, PINK1 stabilizes on the outer mitochondrial membrane (OMM) and recruits Parkin. After recruitment, PINK1 phosphorylates Parkin at Ser65, thus activating Parkin which promotes the ubiquitination of OMM proteins resulting in the subsequent lysosomal degradation of the mitochondria. Previously, we reported that tumor necrosis factor α (TNFα), a proinflammatory cytokine produced during inflammatory airway diseases, increases force generation and ATP hydrolysis, and results in increased mitochondrial fragmentation in human airway smooth muscle (hASM) cells. We also showed that TNFα induces PGC1α signaling, thus promoting mitochondrial biogenesis and increased mitochondrial volume density. In the present study, we hypothesize that TNFα induces mitophagy to promote selective clearance of dysfunctional mitochondria, thus maintaining a healthy mitochondrial pool to meet the increased energy demand in hASM cells. Experiments were performed in hASM cells dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors with no current history of smoking or respiratory diseases. The hASM cells were serum deprived for 48 h and the phenotype was confirmed prior to experimentation. hASM cells were separated into three 6-h treatment groups: 1) TNFα (20 ng/mL), 2) FCCP (5 μM; positive control), and 3) untreated controls. The mitochondria and lysosomes in hASM cells were labelled with MitoTracker green (200 nM) and LysoTracker red (100 nM), respectively and imaged in 3D (Z optical slice of 0.5 μm) using an oil-immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The 3D images were used to determine the coefficient for colocalization of mitochondria with lysosomes, lysosomal volume density, mitochondrial volume density, and mitochondrial complexity index (MCI). Protein expression and phosphorylation status of PINK1, Parkin, phospho-Parkin, VDAC (voltage-dependent anion channel), and LAMP1 (Lysosomal-associated membrane protein 1) were quantified by western blot. We observed that TNFα and FCCP treatment increased lysosomal volume density and mitochondrial colocalization with lysosomes in hASM cells, indicating the formation of mitophagosomes, characteristic of mitophagy. Consistent with previous findings, TNFα treatment increased mitochondrial volume density and decreased MCI, indicative of increased mitochondrial fragmentation. In addition, TNFα treatment increased the expression and mitochondrial translocation of PINK1 and Parkin. The results suggest that TNFα activates PINK1/Parkin-mediated mitophagy in hASM cells. Supported by NIH grants HL157984 (GCS) and HL105355 (OARR) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

RVM-GSM: Classification of OCT Images of Genitourinary Syndrome of Menopause Based on Integrated Model of Local-Global Information Pattern.

Genitourinary syndrome of menopause (GSM) is a group of syndromes, including atrophy of the reproductive tract and urinary tract, and sexual dysfunction, caused by decreased levels of hormones, such as estrogen, in women during the transition to, or late stage of, menopause. GSM symptoms can gradually become severe with age and menopausal time, seriously affecting the safety, and physical and mental health, of patients. Optical coherence tomography (OCT) systems can obtain images similar to "optical slices" in a non-destructive manner. This paper presents a neural network, called RVM-GSM, to implement automatic classification tasks for different types of GSM-OCT images. The RVM-GSM module uses a convolutional neural network (CNN) and a vision transformer (ViT) to capture local and global features of the GSM-OCT images, respectively, and, then, fuses the two features in a multi-layer perception module to classify the image. In accordance with the practical needs of clinical work, lightweight post-processing is added to the final surface of the RVM-GSM module to compress the module. Experimental results showed that the accuracy rate of RVM-GSM in the GSM-OCT image classification task was 98.2%. This result is better than those of the CNN and Vit models, demonstrating the promise and potential of the application of RVM-GSM in the physical health and hygiene fields for women.

Time-domain full-field optical coherence tomography (TD-FF-OCT) in ophthalmic imaging.

Ocular imaging plays an irreplaceable role in the evaluation of eye diseases. Developing cellular-resolution ophthalmic imaging technique for more accurate and effective diagnosis and pathogenesis analysis of ocular diseases is a hot topic in the cross-cutting areas of ophthalmology and imaging. Currently, ocular imaging with traditional optical coherence tomography (OCT) is limited in lateral resolution and thus can hardly resolve cellular structures. Conventional OCT technology obtains ultra-high resolution at the expense of a certain imaging range and cannot achieve full field of view imaging. In the early years, Time-domain full-field OCT (TD-FF-OCT) has been mainly used for ex vivo ophthalmic tissue studies, limited by the low speed and low full-well capacity of existing two-dimensional (2D) cameras. The recent improvements in system design opened new imaging possibilities for in vivo applications thanks to its distinctive optical properties of TD-FF-OCT such as a spatial resolution almost insensitive to aberrations, and the possibility to control the curvature of the optical slice. This review also attempts to look at the future directions of TD-FF-OCT evolution, for example, the potential transfer of the functional-imaging dynamic TD-FF-OCT from the ex vivo into in vivo use and its expected benefit in basic and clinical ophthalmic research. Through non-invasive, wide-field, and cellular-resolution imaging, TD-FF-OCT has great potential to be the next-generation imaging modality to improve our understanding of human eye physiology and pathology.

SDN-based band-adaptive quality assurance scheme in support of heterogenous B5G services over sliceable multi-band optical networks

The rise of traffic intensive services and applications is pushing the limits of conventional single band Wavelength Division Multiplexing (WDM) optical networks. As an answer to this challenge, new data plane technologies are being investigated. Multi-band optical networks have raised as a very interesting candidate due to the potential increased capacity they offer thanks to the exploitation of multiple bands of the optical spectrum. Considering the whole telecom ecosystem, multi-band optical networks will coexist with other technological segments (e.g., Radio Access Network (RAN)) with the aim of provisioning services across the end-to-end infrastructure. With the advent of 5G and beyond 5G (B5G) architectures, novel provisioning paradigms are taking preponderance, such as the case of network slicing, which represents a radical paradigm change with respect to legacy business and provisioning models. As such, proper solutions for supporting network slice provisioning and runtime maintenance at the data plane are required. With this in mind, in this paper we present a control and orchestration architecture for the configuration and maintenance of network slices in multi-band optical networks, in support of B5G end-to-end services. Indeed, quality assurance and maintenance at all levels is seen as a cornerstone in B5G architectures. Thus, proper mechanisms adapted to the nature of the underlying sliceable multi-band data plane are required to ensure the quality of deployed slices. In this regard, we also present a novel band-adaptive protection scheme which takes advantage of the properties of the multi-band data plane so as to enhance the robustness of slices against quality degradations. We showcase the provisioning and maintenance of multi-band optical network slices by means of an experimental demonstration in a real testbed deployed at our premises. In addition, we evaluate the performance of the proposed band-adaptive protection scheme for slice quality assurance in front of other strategies by means of extensive simulation analysis in larger network scenarios.

Applying Deep Learning Cancer Subtyping Algorithms Trained on Physical Slides to Multiphoton Imaging of Unembedded Samples

Abstract Introduction/Objective Deep learning algorithms on digital images of physical tissue slides have shown potential improvements in accuracy and precision of diagnostic interpretation of neoplastic histology. Clustering-constrained- attention multiple-instance learning (CLAM) is one such method that identifies diagnostic sub-regions to accurately classify whole slides. Often, algorithm performance degrades when deployed on datasets that differ from the original set and it is subject to physical slide preparation variability. New multiphoton imaging modalities have potential workflow and quality advantages over physical slides, producing images analogous to whole slide imaging (WSI) without cutting artifacts, but performance of existing algorithms trained on digitized physical slides and applied to multiphoton images remains completely unknown. Given this, we aimed to test the performance of CLAM algorithms for subtyping renal cell carcinoma (RCC) and lung cancer (LC) applied to pseudo-colored multiphoton WSI. Methods/Case Report Clinical RCC and LC surgical resection samples were processed and imaged by Clearing Histology with MulitPhoton microscopy (CHiMP, Applikate Technologies, Fairfield, CT), producing digital images of un- cut, un-embeded tissue to generate H&amp;E-like optical slices. Multiphoton images were downscaled to 0.5 um/px to match algorithm target resolution. CLAM models for subtyping RCC (chromophobe, clear cell, papillary) and LC (squamous &amp; adenocarcinoma) previously trained using TCGA and CPTAC whole slide images of physical slides were applied directly to CHiMP multiphoton images without adjustment. Reference cancer subtype classifications were provided from physical and digital slides. Results (if a Case Study enter NA) For the subtypes included during training, multiphoton WSIs of RCC and LC were accurately subtyped by the CLAM models without stain normalization nor network fine tuning producing high prediction levels. Subtypes not included during the training (namely oncocytoma for RCC) resulted in low scoring model predictions (below 0.85), indicating specificity of identification. Multiple slide levels improved interpretation of several difficult cases for CLAM predictions. Conclusion This preliminary data suggests that CLAM models trained on standard H&amp;E WSIs for RCC and LC subtyping are applicable to pseudo-H&amp;E multiphoton WSIs without domain adaptations. This implies that diagnostic histologic features have been learned by these CLAM models and are efficiently recognized in digital histology images produced via CHiMP.

Pitfalls in methods to study colocalization of nanoparticles in mouse macrophage lysosomes

BackgroundIn the field of nanoscience there is an increasing interest to follow dynamics of nanoparticles (NP) in cells with an emphasis on endo-lysosomal pathways and long-term NP fate. During our research on this topic, we encountered several pitfalls, which can bias the experimental outcome. We address some of these pitfalls and suggest possible solutions. The accuracy of fluorescence microscopy methods has an important role in obtaining insights into NP interactions with lysosomes at the single cell level including quantification of NP uptake in a specific cell type.MethodsHere we use J774A.1 cells as a model for professional phagocytes. We expose them to fluorescently-labelled amorphous silica NP with different sizes and quantify the colocalization of fluorescently-labelled NP with lysosomes over time. We focus on confocal laser scanning microscopy (CLSM) to obtain 3D spatial information and follow live cell imaging to study NP colocalization with lysosomes.ResultsWe evaluate different experimental parameters that can bias the colocalization coefficients (i.e., Pearson’s and Manders’), such as the interference of phenol red in the cell culture medium with the fluorescence intensity and image post-processing (effect of spatial resolution, optical slice thickness, pixel saturation and bit depth). Additionally, we determine the correlation coefficients for NP entering the lysosomes under four different experimental set-ups. First, we found out that not only Pearson’s, but also Manders’ correlation coefficient should be considered in lysosome-NP colocalization studies; second, there is a difference in NP colocalization when using NP of different sizes and fluorescence dyes and last, the correlation coefficients might change depending on live-cell and fixed-cell imaging set-up.ConclusionsThe results summarize detailed steps and recommendations for the experimental design, staining, sample preparation and imaging to improve the reproducibility of colocalization studies between the NP and lysosomes.Graphical

Simulation of Calcium Dynamics in Realistic Three-Dimensional Domains.

The cytosolic concentration of free calcium ions () is an important intracellular messenger in most cell types, and the spatial distribution of is often critical. In a salivary gland acinar cell, a polarised epithelial cell, whose principal function is to transport water and thus secrete saliva, controls the secretion of primary saliva, but increases in are localised to the apical regions of the cell. Hence, any quantitative explanation of how controls saliva secretion must take into careful account the spatial distribution of the various sources, sinks, and -sensitive ion channels. Based on optical slices, we have previously constructed anatomically accurate three-dimensional models of seven salivary gland acinar cells, and thus shown that a model in which responses are confined to the apical regions of the cell is sufficient to provide a quantitative and predictive explanation of primary saliva secretion. However, reconstruction of such anatomically accurate cells is extremely time consuming and inefficient. Here, we present an alternative, mostly automated method of constructing three-dimensional cells that are approximately anatomically accurate and show that the new construction preserves the quantitative accuracy of the model.

OSDL: Dedicated optical slice provisioning in support of distributed deep learning

Networks are the well-known bottlenecks for distributed deep learning (DDL) jobs. The DDL jobs require topologies matching their communication patterns as well as high and stable bandwidth in network transmission, which makes the traditional optimization methods in the field of electrical packet switching (EPS) perform poorly when adapting to the DDL jobs. In contrast, optical circuit switching (OCS) has the natural advantages of topology reconstruction and high bandwidth, which can well fulfill the network requirements and reduce the in-network delay of the DDL jobs. Consequently, in the present work, we propose Optical Slicing provisioning in support of DDL jobs (OSDL). Practically, over an EPS + OCS hybrid network, to match the network topology and the traffic patterns of DDL jobs, we comprehensively design and propose corresponding algorithms for job placement, and scheduling of both OCS and EPS. We first evaluate OSDL with large-scale networks and high-density DDL jobs, by simulations. The simulation results show that OSDL outperforms multiple well-known scheduling methods. More specifically, the speedup of OSDL achieves up to 3.31 times and 10.48 times, over a homogeneous network and a heterogeneous network, respectively. Additionally, we conduct experiments with a relatively small-scale network and relatively low-density DDL jobs, we still observe up to 1.74 times of the speedup. The simulations and the experiments verify the effectiveness of OSDL in accelerating DDL jobs.

Fiber Type Differences in SDHmax and Mitochondrial Volume Density between Diaphragm and Tibialis Anterior Muscles

The diaphragm (DIAm) and tibialis anterior (TA) muscles comprise both fatigue resistant (type I and IIa) and fatigable (type IIx/IIb) muscle fibers. Type I and IIa fibers in the DIAm are very frequently recruited for breathing, whereas in the TA, type I and IIa fibers are recruited for balance and locomotion. More fatigable type IIx/IIb fibers are only infrequently recruited for higher force behaviors in both the DIAm and TA. The repetitive activation of the DIAm during breathing is associated with a duty cycle of 40‐50%, making the DIAm one of, if not the most active muscle in the body ‐ much more active than the TA. These differences in activation history lead to differences in energetic demands between the DIAm and TA. We hypothesized that type I and IIa fibers in the DIAm exhibit a greater mitochondrial volume density and respiratory capacity (SDHmax) compared to type I and IIa fibers in the TA. Muscle segments from both DIAm and TA were dissected from adult female and male Sprague‐Dawley rats (~300 g), pinned to cork at optimal sarcomere length and rapidly frozen in melting isopentane. Subsequently, transverse serial sections of muscle fibers were cut at 10 µm thickness. In a series of transverse sections, muscle fiber types were determined by immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. In an alternate series of sections of the same muscle fibers, mitochondria were labeled using MitoTracker Green. In a third series of alternate sections, the maximum velocity of the succinate dehydrogenase reaction (SDHmax) was measured using a quantitative histochemical assay in which accumulation of the reaction product, nitroblue tetrazolium diformazan (NBTdfz), was measured as a change in optical density (OD) in muscle fibers every 15 s for a 10‐min period. Muscle fiber sections were imaged in 3D using an Olympus Fluoview confocal microscope (40X oil immersion lens; NA 1.4) with an optical slice of 0.5 µm. In the mitoTracker labeled sections, each optical slice was deconvolved to increase spatial resolution (0.125 µm) and after thresholding, binarized images were reconstructed in 3D and analyzed for mitochondrial volume and mitochondrial complexity index (MCI= SA3/16µ2V2) using ImageJ and NIS elements. Mitochondrial volume density and morphometry were analyzed using ImageJ. The mitochondrial volume density and SDHmax were greater in type I and IIa fibers of the DIAm compared to the TA. On the other hand, mitochondrial volume density and SDHmax of type IIx/IIb fibers were comparable between the DIAm and TA. These results support mitochondrial structural and functional adaptations in type I and IIa DIAm fibers based on activation history and greater energetic demand.

Aging Selectively Reduces Mitochondrial Volume Density and Respiratory Capacity in Type IIx/IIb Diaphragm Muscle Fibers

Diaphragm muscle (DIAm) sarcopenia involves selective atrophy of type IIx/IIb DIAm fibers. In addition, there is an age‐related decrease in the maximum velocity of the succinate dehydrogenase reaction (SDHmax) in type IIx/IIb fibers. SDH is a key enzyme of the tricarboxylic acid (TCA) cycle as well as complex II of the electron transport chain (ETC). These results suggest that sarcopenia may reduce mitochondrial volume density in type IIx/IIb DIAm fibers and thereby reduce SDHmax. Alternatively, sarcopenia may reduce the intrinsic respiratory capacity of mitochondria in type IIx/IIb DIAm fibers. We hypothesized that there is an age‐related selective reduction in mitochondrial volume density and SDHmax per mitochondrial volume in type IIx/IIb DIAm fibers. In 24 Fischer 344 rats (6‐mo: n=6 female, n=6 male; 24‐mo: n=6 female, n=6 male), DIAm samples were dissected from the mid‐costal region and rapidly frozen at optimal sarcomere length. In 10 mm cross‐sections, DIAm fiber types were determined by immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. Mitochondria were labelled using Mitotracker Green and imaged using 3D confocal microscopy (40X oil immersion lens; NA 1.4) with an optical slice of 0.5 µm. Each optical slice was deconvolved to increase spatial resolution (0.125 µm) and after thresholding, binarized images were reconstructed in 3D and analyzed for mitochondrial volume and mitochondrial complexity index (MCI=SA3/16µ2V2) using ImageJ and NIS elements. In alternate 6 mm sections, SDHmax was measured using a quantitative histochemical assay in which accumulation of nitro blue tetrazolium diformazan (NBTdfz) was measured in DIAm fibers every 15 s for 10 min. Consistent with previous findings, type IIx/IIb DIAm fibers were ~36% smaller in 24‐mo compared to 6‐mo rats (P&lt;0.0001), whereas there were no age‐related differences in cross‐sectional areas (CSA) of type I and IIa fibers. Mitochondrial volume density was lower in IIx/IIb fibers of 24‐mo compared to 6‐mo rats (P&lt;0.0001), whereas there were no age‐related differences in type I and IIa fibers. The MCI in type IIx/IIb fibers was lower (greater fragmentation) in 24‐mo compared to 6‐mo rats (P&lt;0.0001), whereas the MCIs of type I and IIa fibers were similar across ages. Finally, SDHmax per fiber volume was lower in type IIx/IIb DIAm fibers in 24‐mo compared with 6‐mo rats (P&lt;0.0001), whereas there were no age‐related differences in type I and IIa fibers. When SDHmax was normalized for mitochondrial volume, there was no age‐related difference in the intrinsic mitochondrial respiratory capacity in type IIx/IIb fibers. Therefore, aging results in reduced mitochondrial volume density and increased fragmentation in type IIx/IIb DIAm fibers, but no change in the intrinsic respiratory capacity of mitochondria.

TNFα Reduces the Maximum Respiratory Capacity of Mitochondria in Human Airway Smooth Muscle Cells

Previously, we reported that airway inflammation mediated by pro‐inflammatory cytokines such as TNFα induces hypercontractility and increased ATP consumption in human airway smooth muscle (hASM). We also found that TNFα induces an increase in maximum O2 consumption rate as well as mitochondrial biogenesis and an increase in mitochondrial volume density. However, when normalized to mitochondrial volume, maximum O2 consumption rate per mitochondrion is reduced in hASM after TNFα exposure. Unfortunately, standard respirometry (e.g., Seahorse) measures only the averaged maximum O2 consumption rate of a large number of cells. In the present study, we used a quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase reaction (SDHmax) together with mitochondrial volume density in individual hASM cells. SDH is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC), and SDHmax reflects the maximum respiratory capacity of individual mitochondrion. We hypothesized that TNFα decreases SDHmax per mitochondrion in hASM cells. hASM cells were dissociated from bronchial biopsies obtained during thoracic surgeries in patients with no history of asthma or chronic obstructive pulmonary disease. The hASM cells were treated with TNFα (20 ng/ml) or vehicle (DMSO) for 24 h, and SDHmax was measured using a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT) as the reaction indicator. As the SDH reaction proceeded, the hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using an oil‐immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The change in optical density (OD), reflecting the accumulation of NBT diformazan reaction product in the hASM cell, was measured every 15 s over a 10 min period. Based on the Beer‐Lambert equation, the slope of the change in OD was used to determine SDHmax expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the same hASM cells were also loaded with MitoTracker Green (500 nM) and imaged in 3D. We found that mitochondria were more fragmented after TNFα exposure although mitochondrial volume density increased. When SDHmax was normalized to mitochondrial volume, we found that SDHmax per mitochondrion decreased in hASM cells, consistent with the reduction in maximum O2 consumption per mitochondrion that we previously observed. Thus, the increased energetic demand induced by inflammation (TNFα) are met by mitochondrial fragmentation leading to biogenesis and an increase in mitochondrial volume density rather than an increase in mitochondrial respiratory capacity.

Single Cell Assessment of Mitochondrial Function

Cellular mitochondrial function can be assessed using high resolution respirometry that measures O2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDHmax) in isolated human airway smooth muscle (hASM) cells using 1‐methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDHmax, the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil‐immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDHmax was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP‐mediated disruption of the mitochondrial proton gradient does not affect SDHmax, while SDHmax is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDHmax was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDHmax can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.