3D Confocal Images Research Articles

Enhancing the textural and rheological properties of fermentation-induced pea protein emulsion gels with transglutaminase.

The aim of this study was to assess how transglutaminase (TG) impacts the microstructure, texture, and rheological properties of fermentation-induced pea protein emulsion gels. Additionally, the study examined the influence of storage time on the functional properties of these gels. Fermentation-induced pea protein gels were produced in the presence or absence of TG and stored for 1, 4, 8, 12, and 16 weeks. Texture analysis, rheological measurements, moisture content and microstructure evaluation with confocal laser scanning microscopy (CLSM) and 3D image analysis were conducted to explore the effects of TG on the structural and rheological properties of the fermented samples. The porosity of the protein networks in the pea gels decreased in the presence of TG, the storage modulus increased and the textural characteristics were significantly improved, resulting in harder and more springy gels. The gel porosity increased in gels with and without TG after storage but the effect of storage on textural and rheological properties was limited, indicating limited structural rearrangement once the fermentation-induced pea protein emulsion gels are formed. Greater coalescence was observed for oil droplets within the gel matrix after 16 weeks of storage in the absence of TG, consistent with these protein structures being weaker than the more structurally stable TG-treated gels. This study shows that TG treatment is a powerful tool to enhance the textural and rheological properties of fermentation-induced pea protein emulsion gels.

Programmable comprehensive controller for multi-color 3D confocal spinning-disk image scanning microscope

This paper introduces a compact, portable, and highly accurate triggering control system for a 3D confocal spinning-disk image scanning microscope (CSD-ISM). Building upon on our previously published research, we expanded the hardware of the controller and synchronized it with a sub-micron translator which scans the object in the z-direction. As well as expanding the hardware, the software also was extended from previously published work similarly as it is stated for hardware while allowing full control over the 3D movement. We showed a clear and smooth 3D image made up of a collection of 2D images at different heights.

Intravenous 3K3A-Activated Protein C Inhibits Murine Ocular Inflammation and Suppresses Ocular Choroidal Neovascularization

Introduction: Choroidal neovascularization (CNV) is characterized by the growth of newly formed abnormal blood vessels from the choroid extending into the neurosensory retina, leading to vision loss. CNV is a common cause of visual impairment in neovascular age-related macular degeneration (nAMD) patients. Current treatments failed to stop the continuity of the neurodegenerative process. Despite the urgency, no neuroprotective agent has been found yet to treat AMD and other neurodegenerative ocular diseases. 3K3A-activated protein C (APC) is a cell-signaling analog of the endogenous blood serine protease APC. Preclinical studies showed that circulating plasma 3K3A-APC readily crosses the blood-brain barrier (BBB) and reaches neurons due to EPCR-dependent transport. In rodent models of stroke, brain injury, and neurodegenerative disorders, 3K3A-APC exerts neuroprotective, anti-inflammatory, and vasculoprotective activities. Due to its pleiotropic effects, 3K3A-APC has advanced to clinical trial stages for treating acute brain damage and chronic neurodegenerative diseases, which share similarities with retinal neurodegenerative disorders. Notably, the retina is part of the central nervous system (CNS), and the blood-retina barrier shares functional and structural similarities with the BBB. We previously demonstrated that intraocular administration of 3K3A-APC functions as a pleiotropic cytoprotective molecule within the retina, reducing ocular inflammation, suppressing CNV growth, and exhibiting promising therapeutic potential as a neuroprotective agent for treating ocular disorders. Intraocular administration is commonly utilized for delivering therapeutics to treat retinal conditions. However, safety concerns, the burden on the healthcare system, and patient comfort may indicate that systemic administration of 3K3A-APC is a beneficial alternative. Aim: To determine whether intravenously injecting 3K3A-APC into mice tails will cross the blood-retina barrier, induce anti-inflammatory activities in the retina, and suppress CNV. Methods: The efficacy of systemic administration of 3K3A-APC was evaluated using two C57BL/6J murine models: the LPS-induced ocular inflammation and the laser-induced CNV model. CNV was triggered by laser and ocular inflammation was triggered by intravitreal injection of lipopolysaccharide (LPS). Murine recombinant 3K3A-APC was administered systemically via tail injection. Fluorescein isothiocyanate (FITC)-dextran perfusion followed by retinal flatmount staining with anti-CD11b was used to assess inflammatory co-localization of cells to retinal vessels. FITC-dextran perfusion followed by retinal pigment epithelium (RPE)-choroid flatmount was used to assess CNV vascular penetration using 3D confocal imaging. Results: LPS injection triggered robust inflammatory cell infiltration in both the anterior and posterior chambers of the eyes. Systemic administration of 3K3A-APC (0.5 mg/kg, 1 and 4 hours post-LPS) significantly inhibited ocular inflammation. Immunostaining performed 24 hours post LPS revealed that 3K3A-APC reduced the number of CD11b+ monocyte and their extravasation into the retinal parenchyma (Figure). In the laser-induced CNV model, systemic administration of 0.2 mg/kg 3K3A-APC was applied one hour and four days post-laser. Evaluation of retinal specimens ten days post-laser showed that 3K3A-APC treatment significantly suppressed CNV growth, attenuated the abnormal growth of blood vessels from the choroid into the sensory retina, and decreased myeloid cell recruitment to the RPE-choroid area. Conclusion: Our study reveals that systemically administered 3K3A-APC effectively inhibits ocular inflammation and reduces CNV formation. Drawing insights from the neuroprotective activities of 3K3A-APC in the brain and CNS and recognizing the retina, particularly the macula, as an extension of the brain, we propose 3K3A-APC as a potential neuroprotective treatment for AMD and other neurodegenerative retinal pathologies. Given the established safety of systemic 3K3A-APC administration in phase 2 clinical trials for ischemic stroke, our findings support further exploring 3K3A-APC as a novel therapeutic approach in ophthalmology. Further research is warranted to elucidate fully the mechanisms underlying 3K3A-APC's beneficial effects on the retina.

Where Do CABs Exist? Verification of a specific region containing concave Actin Bundles (CABs) in a 3-Dimensional confocal image.

CABs (Concave Actin Bundles) are oriented against the scaffold transversally in a manner different from traditional longitudinal F-actin bundles. CABs are present in a specific area, and do not exist in random areas. Biologically, CABs are developed to attach cells to fibers firmly so that CABs are found near cells. Based on this knowledge, we closely examined 3D confocal microcopy images containing fiber scaffolds, actin, and cells. Then, we assumed that the areas containing high values of compactness of fiber, compactness of actin, and density of cells would have many numbers of CABs.In this research, we wanted to prove this assumption. We first incorporated a two-point correlation function to define a measure of compactness. Then, we used the Bayes' theorem to prove the above assumption. As the assumption, our results verified that CABs exist in an area of high compactness of a fiber network, high compactness of actin distribution, and high density of cells. Thus, we concluded that CABs are developed to attach cells to a fibrillar scaffold firmly. This finding may be further verified mathematically in future studies.

Impact of mucus modulation by N-acetylcysteine on nanoparticle toxicity

Human respiratory mucus is a biological hydrogel that forms a protective barrier for the underlying epithelium. Modulation of the mucus layer has been employed as a strategy to enhance transmucosal drug carrier transport. However, a drawback of this strategy is a potential reduction of the mucus barrier properties, in particular in situations with an increased exposure to particles. In this study, we investigated the impact of mucus modulation on its protective role. In vitro mucus was produced by Calu-3 cells, cultivated at the air-liquid interface for 21 days and used for further testing as formed on top of the cells. Analysis of confocal 3D imaging data revealed that after 21 days Calu-3 cells secrete a mucus layer with a thickness of 24 ± 6 μm. Mucus appeared to restrict penetration of 500 nm carboxyl-modified polystyrene particles to the upper 5–10 μm of the layer. Furthermore, a mucus modulation protocol using aerosolized N-acetylcysteine (NAC) was developed. This treatment enhanced the penetration of particles through the mucus down to deeper layers by means of the mucolytic action of NAC. These findings were supported by cytotoxicity data, indicating that intact mucus protects the underlying epithelium from particle-induced effects on membrane integrity. The impact of NAC treatment on the protective properties of mucus was probed by using 50 and 100 nm amine-modified and 50 nm carboxyl-modified polystyrene nanoparticles, respectively. Cytotoxicity was only induced by the amine-modified particles in combination with NAC treatment, implying a reduced protective function of modulated mucus. Overall, our data emphasize the importance of integrating an assessment of the protective function of mucus into the development of therapy approaches involving mucus modulation.

Methods for dynamic and whole volume imaging of the zebrafish heart

Tissue development and regeneration are dynamic processes involving complex cell migration and cell-cell interactions. We have developed a protocol for complementary time-lapse and three-dimensional (3D) imaging of tissue for developmental and regeneration studies which we apply here to the zebrafish cardiac vasculature. 3D imaging of fixed specimens is used to first define the subject at high resolution then live imaging captures how it changes dynamically. Hearts from adult and juvenile zebrafish are extracted and cleaned in preparation for the different imaging modalities. For whole-mount 3D confocal imaging, single or multiple hearts with native fluorescence or immuno-labeling are prepared for stabilization or clearing, and then imaged. For live imaging, hearts are placed in a prefabricated fluidic device and set on a temperature-controlled microscope for culture and imaging over several days. This protocol allows complete visualization of morphogenic processes in a 3D context and provides the ability to follow cell behaviors to complement in vivo and fixed tissue studies. This culture and imaging protocol can be applied to different cell and tissue types. Here, we have used it to observe zebrafish coronary vasculature and the migration of coronary endothelial cells during heart regeneration.

Design of Water-Soluble Rotaxane-Capped Superparamagnetic, Ultrasmall Fe3O4 Nanoparticles for Targeted NIR Fluorescence Imaging in Combination with Magnetic Resonance Imaging.

Integrating an NIR fluorescent probe with a magnetic resonance imaging (MRI) agent to harvest complementary imaging information is challenging. Here, we have designed water-soluble, biocompatible, noncytotoxic, bright-NIR-emitting, sugar-functionalized, mechanically interlocked molecules (MIMs)-capped superparamagnetic ultrasmall Fe3O4 NPs for targeted multimodal imaging. Dual-functional stoppers containing an unsymmetrical NIR squaraine dye interlocked within a macrocycle to construct multifunctional MIMs are developed with enhanced NIR fluorescence efficiency and durability. One of the stoppers of the axle is composed of a lipophilic cationic TPP+ functionality to target mitochondria, and the other stopper comprises a dopamine-containing catechol group to anchor at the surface of the synthesized Fe3O4 NPs. Fe3O4 NPs surface-coated with targeted NIR rotaxanes help to deliver ultrasmall magnetic NPs specifically inside the mitochondria. Two carbohydrate moieties are conjugated with the macrocycle of the rotaxane via click chemistry to improve the water solubility of MitoSQRot-(Carb-OH)2-DOPA-Fe3O4 NPs. Water-soluble, rotaxane-capped Fe3O4 NPs are used for live-cell mitochondria-targeted NIR fluorescence confocal imaging, 3D and multicolor imaging in combination with T2-weighted MRI on a 9.4 T MR scanner with a high relaxation rate (r2) of 180.7 mM-1 s-1. Biocompatible, noncytotoxic, ultrabright NIR rotaxane-capped superparamagnetic ultrasmall monodisperse Fe3O4 NPs could be a promising agent for targeted multimodal imaging applications.

Use of artificial neural networks to identify and analyze polymerized actin‐based cytoskeletal structures in 3D confocal images

We propose an artificial neural network (ANN) as a kernel function of the recognizer of legitimate CABs from candidate CABs, that does not need human interventions. The performance of the recognizer shows noticeable recognition accuracy and addresses shortcomings of previous methods, including the need for human visual validation to recognize CABs from candidate CABs. Further, it helps to find and reduce errors resulting from human visual validation, which in turn would provide biologists/biophysicists a more comprehensive. understanding of a CAB.BackgroundLiving cells need to undergo subtle shape adaptations in response to the topography of their substrates. These shape changes are mainly determined by reorganization of their internal cytoskeleton, with a major contribution from filamentous (F) actin. Bundles of F‐actin play a major role in determining cell shape and their interaction with substrates, either as “stress fibers,” or as our newly discovered “Concave Actin Bundles” (CABs), which mainly occur while endothelial cells wrap micro‐fibers in culture.MethodsTo better understand the morphology and functions of these CABs, it is necessary to recognize and analyze as many of them as possible in complex cellular ensembles, which is a demanding and time‐consuming task. In this study, we present a novel algorithm to automatically recognize CABs without further human intervention. We developed and employed a multilayer perceptron artificial neural network (“the recognizer”), which was trained to identify CABs.ResultsThe recognizer demonstrated high overall recognition rate and reliability in both randomized training, and in subsequent testing experiments.ConclusionIt would be an effective replacement for validation by visual detection which is both tedious and inherently prone to errors.

Postnatal development of extracellular matrix and vascular function in small arteries of the rat.

Introduction: Vascular extracellular matrix (ECM) is dominated by elastic fibers (elastin with fibrillin-rich microfibrils) and collagens. Current understanding of ECM protein development largely comes from studies of conduit vessels (e.g., aorta) while resistance vessel data are sparse. With an emphasis on elastin, we examined whether changes in postnatal expression of arteriolar wall ECM would correlate with development of local vasoregulatory mechanisms such as the myogenic response and endothelium-dependent dilation. Methods: Rat cerebral and mesenteric arteries were isolated at ages 3, 7, 11, 14, 19days, 2months, and 2years. Using qPCR mRNA expression patterns were examined for elastin, collagen types I, II, III, IV, fibrillin-1, and -2, lysyl oxidase (LOX), and transglutaminase 2. Results: Elastin, LOX and fibrillar collagens I and III mRNA peaked at day 11-14 in both vasculatures before declining at later time-points. 3D confocal imaging for elastin showed continuous remodeling in the adventitia and the internal elastic lamina for both cerebral and mesenteric vessels. Myogenic responsiveness in cannulated cerebral arteries was detectable at day 3 with constriction shifted to higher intraluminal pressures by day 19. Myogenic responsiveness of mesenteric vessels appeared fully developed by day 3. Functional studies were performed to investigate developmental changes in endothelial-dependent dilation. Endothelial-dependent dilation to acetylcholine was less at day 3 compared to day 19 and at day 3 lacked an endothelial-derived hyperpolarizing factor component that was evident at day 19. Conclusion: Collectively, in the rat small artery structural remodeling and aspects of functional control continue to develop in the immediate postnatal period.

Maternal high-fat diet increases airway sensory innervation and reflex bronchoconstriction in adult offspring.

Children born to obese mothers are prone to develop asthma and airway hyperresponsiveness, but the mechanisms behind this are unclear. Here we developed a mouse model of maternal diet-induced obesity that recapitulates metabolic abnormalities seen in humans born to obese mothers. Offspring of dams fed a high-fat diet (HFD) showed increased adiposity, hyperinsulinemia, and insulin resistance at 16 wk of age despite being fed only a regular diet (RD). Bronchoconstriction induced by inhaled 5-hydroxytriptamine was also significantly increased in offspring of HFD-fed versus RD-fed dams. Increased bronchoconstriction was blocked by vagotomy, indicating this reflex was mediated by airway nerves. Three-dimensional (3-D) confocal imaging of tracheas collected from 16-wk-old offspring showed that both epithelial sensory innervation and substance P expression were increased in the offspring of HFD-fed dams compared with offspring of RD-fed dams. For the first time, we show that maternal high-fat diet increases airway sensory innervation in offspring, leading to reflex airway hyperresponsiveness.NEW & NOTEWORTHY Our study reveals a novel potential mechanism, by which maternal high-fat diet increases the risk and severity of asthma in offspring. We found that exposure to maternal high-fat diet in mice leads to hyperinnervation of airway sensory nerves and increased reflex bronchoconstriction in offspring fed a regular diet only. These findings have important clinical implications and provide new insights into the pathophysiology of asthma, highlighting the need for preventive strategies in this patient population.

A high temporal-spatial resolution three-dimensional imaging method using arrayed-focus structured illumination with differential axial enhancement

High precision and fast three-dimensional (3D) microscopic imaging technology plays an extremely important role in MEMS, micro optics, microelectronics, biomedicine and other fields. In this paper, a confocal microscopic 3D imaging method based on differential array focusing structured light illumination (DAF-SIM) is proposed. Theoretical analysis and experimental results show that under the 100x/NA 0.95 objective lens, the lateral resolution of this method reaches 173 nm, exceeding the optical diffraction limit, the axial sensitivity is better than 10 nm, and the time resolution is about 3 Hz@2.3 M Pixel, achieving the goal of high horizontal and axial resolution and time resolution. The high spatial and temporal resolution of DAF-SIM 3D micro imaging method provides a highly applicable, efficient and high-precision online micro terrain detection scheme for intelligent manufacturing and other fields.

An advanced organic molecular probe for multimodal fluorescence imaging of cellular lipid droplets

Lipid droplets (LDs) are critical cellular organelles associated with various physiological processes in eukaryotic cells. To visualize LDs and research their complex physiological functions, various fluorescence imaging models including the confocal imaging, the lambda scan imaging and the stimulated emission depletion (STED) super-resolution imaging have been successfully employed in recent years. However, since different fluorescence imaging models have different requirements for fluorescent probes, it is very challenging to employ single fluorescent probe for multimodal fluorescence imaging. Herein, an advanced organic fluorescent probe Lipi-DSBOMe has been sophisticatedly developed for purpose. Featuring with the advantages of high staining selectivity towards LDs, polarity sensitive luminescence, high photostability and low STED saturation intensity, this fluorescent probe is well suited for multimodal fluorescence imaging. For instance, employed with this probe for three-dimensional (3D) confocal imaging to quantitatively determine the change of number of LDs under the oleic acid stimulation, lambda scan imaging to precisely measure the polarity of LDs in different types of cells, and STED imaging to measure the size of LDs with the state-of-the-art resolution (42 nm). In overall, employing this single fluorescent probe for multimodal fluorescence imaging has allowed to provide abundant information about LDs, thus effectively promoting the LDs biology studies.

Abstract 4557: 3D-EXpress platform utilizing tumoroids from patients with MSS and MSI-H tumors allows rapid assessment of anti-tumor activity of immune checkpoint inhibitors and development of clinically relevant biomarkers of treatment response

Abstract Introduction: Recent studies showed that patients with deficient mismatch repair(dMMR)/microsatellite instability-high (MSI-H) tumors, have higher sensitivity to immune checkpointinhibitors (ICIs) compared to patients with microsatellite-stable (MSS)/microsatellite instability-low(MSI-L) tumors. However, the mechanisms of treatment responsiveness and resistance is not wellunderstood. Here, we used a novel 3D-EXpress ex vivo fresh patient tumoroid platform to assess theefficacy of nivolumab/ipilimumab combination therapy in tumors with known MSS/MSI-H status andperformed correlative studies. Materials and Methods: All tumor samples were obtained with patient consent and relevant IRBapproval. Tumoroids measuring 150 µm in size retaining tumor cell heterogeneity, tumor-residentimmune cells, stromal components, and cell-extracellular matrix interaction were prepared frompatients with endometrial and colorectal tumors among others. Tumoroid aliquots were cryopreservedin Nilogen’s tumoroid Biorepository for future studies. For the 3D-EXpress studies cryopreservedtumoroids were selected based on MSS/MSI-H status and treated ex vivo with nivolumab andipilimumab alone and in combinations for 72h. Tumor responses to treatments were evaluated by aproprietary tumor cell killing assay and changes in tumor immune microenvironment. Furthermore,tumor PD-L1 expression levels were analyzed on the associated TMA slides. Results: Treatment-induced tumor cell killing activity in intact tumoroids was assessed by a 3D high-content confocal imaging technique using a proprietary algorithm for data analysis. The impact of exvivo treatment by nivolumab and/or ipilimumab on tumor resident immune cell populations wasmonitored by a multiplex cytokine release assay analyzing release of GM-CSF, sCD137, IFNγ, sFas, sFasL,Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α, and Perforin usingculture supernatants isolated from treated tumoroid samples. Based on the ex vivo responses tumorswere assigned to treatment sensitive and resistant groups and correlative analyses were performed withindividual tumors’ innate and adaptive immune cell populations detected by a 21-color flow cytometrypanel, in addition to tumor MSS/MSI-H and p53 status, and detailed clinicopathologic data readilyavailable for the tumors cryopreserved in the Biorepository. Conclusion: The 3D-Express platform, using cryopreserved 3D tumoroids with intact TME is an effectivetool for the assessment of rational combinations which may prove relevant in the treatment of solidtumors. Furthermore, we believe this platform is a useful tool for the pre-clinical assessment of specifictherapeutic regimens designed for individualized patient care. Citation Format: Brittney Ruedlinger, Seth Currlin, Sharon Camacho, Alliyah Humphrey, Jared Ehrhart, Soner Altiok. 3D-EXpress platform utilizing tumoroids from patients with MSS and MSI-H tumors allows rapid assessment of anti-tumor activity of immune checkpoint inhibitors and development of clinically relevant biomarkers of treatment response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4557.

Abstract 206: Organoid models for predicting drug response in high grade serous and triple negative breast cancer

Abstract While most patients with high grade serous ovarian cancer (HGSC) respond to platinum-based chemotherapy, the response is rarely durable and recurrence almost inevitable. Women with triple negative breast cancer (TNBC) urgently require effective therapeutic options. In women with HGSC and TNBC (with mutations in BRCA1/2) treatment with a Poly-ADP-ribose inhibitor (PARPi) has emerged as a standard of care. In models of HGSC and TNBC senescence may play a role in PARPi resistance with i Bcl-xL, a member of the Bcl-2 family of proteins, preventing apoptosis. Therefore, we have initiated a clinical trial whereby a PARPi and then, an inhibitor of Bcl-xL, Navitoclax, will be added to their course of treatment [NCT05358639]. Bcl-xL is only one of the five known inhibitors of apoptosis, at present we do not know in vivo how these will impact the efficacy of the PARPi/Navitoclax combination or if they will provide other targets for effective therapy. Our hypothesis is that patient derived organoids can be used as a pragmatic way to assess the efficacy of new drugs and to identify for individual patients which drug(s) and drug combinations are likely to be most efficacious. In particular, with reference to the clinical trial, identify for individual patients which Bcl-2 protein inhibitor will synergize with a PARPi to optimize treatment. Currently we are using organoids to test novel drugs and drug combinations and to develop biomarkers for HGSC and TNBC treatment response. A limitation to the use of organoids generated from biopsy samples is the lack of efficient and reliable experimental models that recapitulate in vitro patient tumors faithfully enough to facilitate translation to therapeutic decisions for patients. By adapting the relatively new technique of conditional reprogramming and combining it with novel hydrogel based synthetic ECM supports we can reproducibly generate HGSC and TNBC patient-specific tumor organoids models in two weeks with greater than 90% success. Organoids grown in 384 or 1536 well format are stained with novel non-toxic dyes and chemoresponses to drugs are inferred from automatically acquired 3D confocal image stacks using deep learning AI algorithms that enable automated analyses. Our data suggest that this approach captures the inherent heterogeneity of the disease, albeit local to the sampled site. We are now employing deep learning AI algorithms to enable automated analyses of 3D confocal image stacks of organoids and infer drug responses that will be compared to patient responses in the clinical trial. Citation Format: Helen J. MacKay, Alla Buzina, Betty Li, Lilian Gien, David Andrews. Organoid models for predicting drug response in high grade serous and triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 206.

3D confocal imaging methodology optimized for pore space characterization of carbonates

Pore space characterization of carbonate materials is of fundamental importance to a wide range of earth science and engineering applications. In this study, we show how confocal microscopy can be used as a reliable tool to visualize and quantify the heterogeneous pore space in carbonate materials. In confocal imagery, the quality of pore space images is controlled by various factors including the choice of fluorophore, objective lens, and medium of imaging. Our experiments demonstrated that there is no “fit-for-all” dye. The red dye provides more depth of investigation, while the blue dye can capture large pores more efficiently. Our investigation indicated that quantitative pore space description from confocal images can be significantly influenced by imaging artifacts associated with imaging the heterogeneous and complex pore space in carbonates. By applying image deconvolution, we mitigated the confocal imaging artifacts (e.g., spherical aberration, and resolution deviation), and extracted a more reliable 3D pore-network model of the carbonate sample. We employed our optimized confocal imaging protocol to visualize and quantify macro- and micropores, and more importantly their interconnectivity within an Indiana limestone (IL) sample. The computed pore-throat size distribution (PSD) from the 3D confocal images was able to capture the inherent bimodal distribution of IL.

Segmentation, tracking, and sub-cellular feature extraction in 3D time-lapse images

This paper presents a method for time-lapse 3D cell analysis. Specifically, we consider the problem of accurately localizing and quantitatively analyzing sub-cellular features, and for tracking individual cells from time-lapse 3D confocal cell image stacks. The heterogeneity of cells and the volume of multi-dimensional images presents a major challenge for fully automated analysis of morphogenesis and development of cells. This paper is motivated by the pavement cell growth process, and building a quantitative morphogenesis model. We propose a deep feature based segmentation method to accurately detect and label each cell region. An adjacency graph based method is used to extract sub-cellular features of the segmented cells. Finally, the robust graph based tracking algorithm using multiple cell features is proposed for associating cells at different time instances. We also demonstrate the generality of our tracking method on C. elegans fluorescent nuclei imagery. Extensive experiment results are provided and demonstrate the robustness of the proposed method. The code is available on GitHub and the method is available as a service through the BisQue portal.

A new organic molecular probe as a powerful tool for fluorescence imaging and biological study of lipid droplets.

Background: Lipid droplets (LDs) are critical organelles associated with many physiological processes in eukaryotic cells. To visualize and study LDs, fluorescence imaging techniques including the confocal imaging as well as the emerging super-resolution imaging of stimulated emission depletion (STED), have been regarded as the most useful methods. However, directly limited by the availability of advanced LDs fluorescent probes, the performances of LDs fluorescence imaging are increasingly unsatisfied with respect to the fast research progress of LDs. Methods: We herein newly developed a superior LDs fluorescent probe named Lipi-QA as a powerful tool for LDs fluorescence imaging and biological study. Colocalization imaging of Lipi-QA and LDs fluorescent probe Ph-Red was conducted in four cell lines. The LDs staining selectivity and the photostability of Lipi-QA were also evaluated by comparing with the commercial LDs probe Nile Red. The in-situ fluorescence lifetime of Lipi-QA in LDs was determined by time-gated detection. The cytotoxicity of Lipi-QA was assessed by MTT assay. The STED saturation intensity as well as the power- and gate time-dependent resolution were tested by Leica SP8 STED super-resolution nanoscopy. The time-lapse 3D confocal imaging and time-lapse STED super-resolution imaging were then designed to study the complex physiological functions of LDs. Results: Featuring with the advantages of the super-photostability, high LDs selectivity, long fluorescence lifetime and low STED saturation intensity, the fluorescent probe Lipi-QA was capable of the long-term time-lapse three-dimensional (3D) confocal imaging to in-situ monitor LDs in 3D space and the time-lapse STED super-resolution imaging (up to 500 STED frames) to track the dynamics of LDs with nanoscale resolution (37 nm). Conclusions: Based on the state-of-the-art fluorescence imaging results, some new biological insights into LDs have been successfully provided. For instance, the long-term time-lapse 3D confocal imaging has surely answered an important and controversial question that the number of LDs would significantly decrease rather than increase upon starvation stimulation; the time-lapse STED super-resolution imaging with the highest resolution has impressively uncovered the fission process of nanoscale LDs for the first time; the starvation-induced change of LDs in size and in speed has been further revealed at nanoscale by the STED super-resolution imaging. All of these results not only highlight the utility of the newly developed fluorescent probe but also significantly promote the biological study of LDs.

A Scalable 3D High-Content Imaging Protocol for Measuring a Drug Induced DNA Damage Response Using Immunofluorescent Subnuclear γH2AX Spots in Patient Derived Ovarian Cancer Organoids.

The high morbidity rate of ovarian cancer has remained unchanged during the past four decades, partly due to a lack of understanding of disease mechanisms and difficulties in developing new targeted therapies. Defective DNA damage detection and repair is one of the hallmarks of cancer cells and is a defining characteristic of ovarian cancer. Most in vitro studies to date involve viability measurements at scale using relevant cancer cell lines; however, the translation to the clinic is often lacking. The use of patient derived organoids is closing that translational gap, yet the 3D nature of organoid cultures presents challenges for assay measurements beyond viability measurements. In particular, high-content imaging has the potential for screening at scale, providing a better understanding of the mechanism of action of drugs or genetic perturbagens. In this study we report a semiautomated and scalable immunofluorescence imaging assay utilizing the development of a 384-well plate based subnuclear staining and clearing protocol and optimization of 3D confocal image analysis for studying DNA damage dose response in human ovarian cancer organoids. The assay was validated in four organoid models and demonstrated a predictable response to etoposide drug treatment with the lowest efficacy observed in the clinically most resistant model. This imaging and analysis method can be applied to other 3D organoid and spheroid models for use in high content screening.

Imaging and plate counting to quantify the effect of an antimicrobial: A case study of a photo-activated chlorine dioxide treatment.

To assess removal versus kill efficacies of antimicrobial treatments against thick biofilms with statistical confidence. A photo-activated chlorine dioxide treatment (Photo ClO2 ) was tested in two independent experiments against thick (>100 μm) Pseudomonas aeruginosa biofilms. Kill efficacy was assessed by viable plate counts. Removal efficacy was assessed by 3D confocal scanning laser microscope imaging (CSLM). Biovolumes were calculated using an image analysis approach that models the penetration limitation of the laser into thick biofilms using Beer's Law. Error bars are provided that account for the spatial correlation of the biofilm's surface. The responsiveness of the biovolumes and plate counts to the increasing contact time of Photo ClO2 were quite different, with a massive 7 log reduction in viable cells (95% confidence interval [CI]: 6.2, 7.9) but a more moderate 73% reduction in biovolume (95% CI: [60%, 100%]). Results are leveraged to quantitatively assess candidate CSLM experimental designs of thick biofilms. Photo ClO2 kills biofilm bacteria but only partially removes the biofilm from the surface. To maximize statistical confidence in assessing removal, imaging experiments should use fewer pixels in each z-slice, and more importantly, at least two independent experiments even if there is only a single field of view in each experiment. There is limited penetration depth when collecting 3D confocal images of thick biofilms. Removal can be assessed by optimally fitting Beer's Law to all of the intensities in a 3D image and by accounting for the spatial correlation of the biofilm's surface. For thick biofilms, other image analysis approaches are biased or do not provide error bars. We generate unbiased estimates of removal and assess candidate CSLM experimental designs of thick biofilms with different pixilations, numbers of fields of view and number of experiments using the included design tool.

Abstract 10279: Three-Dimensional Deep-Tissue Imaging of the Right Ventricle Reveals the Complex Remodeling of the Microvascular Network in Right Heart Failure

Introduction: Right ventricular (RV) failure is the leading cause of death in patients with pulmonary arterial hypertension (PAH). Capillary rarefaction has been proposed as the hallmark of RV failure, yet our understanding of the capillary architecture is limited by 2D image analysis. Three-dimensional (3D) confocal deep tissue imaging offers the unparalleled opportunity to characterize the architectural microvascular changes in relationship to cardiomyocytes in pressure-overloaded RV failure. Methods and Results: 250 μm heart tissue slices from mice with RV failure, 7-weeks after Pulmonary Artery Banding (PAB) were harvested and the microvascular network was assessed for density, segment length, diameter, orientation, as well as microvasculature-cardiomyocyte contact area in regions with and without interstitial fibrosis using confocal 3D imaging. Human heart tissue from end-stage PAH patients was compared to controls with normal RV function. In PAB mice, 3D imaging of the RV free wall revealed tortuous, shorter, thicker and higher-branched microvascular segments as well as development of fibrosis compared to Sham animals (Fig A). When corrected for fibrosis, the microvascular density was preserved even in advanced stages of RV failure. Physical contact between the microvasculature and cardiomyocytes was preserved in areas without fibrosis, yet significantly impaired in areas with interstitial fibrosis (Fig B), accompanied by an overexpression of β-myosin heavy chain suggesting local ischemia (Fig C). 3D imaging of human RV tissue of end-stage PAH patients revealed architectural microvascular remodeling that varied depending on etiologies and length of disease yet showed preserved microvascular density. (Fig. D). Conclusions: 3D deep tissue imaging revealed compensatory changes of the microvascular network to maintain a stable cardiomyocyte coverage in pressure-overloaded RV failure, which was impaired in areas of interstitial fibrosis.