Three-dimensional Confocal Imaging Research Articles

Novel High-Resolution Three-Dimensional Fluorescent Confocal Imaging Resolves Differences in Urinary Sediment

Novel High-Resolution Three-Dimensional Fluorescent Confocal Imaging Resolves Differences in Urinary Sediment

Harnessing forward multiple scattering for optical imaging deep inside an opaque medium

As light travels through a disordered medium such as biological tissues, it undergoes multiple scattering events. This phenomenon is detrimental to in-depth optical microscopy, as it causes a drastic degradation of contrast, resolution and brightness of the resulting image beyond a few scattering mean free paths. However, the information about the inner reflectivity of the sample is not lost; only scrambled. To recover this information, a matrix approach of optical imaging can be fruitful. Here, we report on a de-scanned measurement of a high-dimension reflection matrix R via low coherence interferometry. Then, we show how a set of independent focusing laws can be extracted for each medium voxel through an iterative multi-scale analysis of wave distortions contained in R. It enables an optimal and local compensation of forward multiple scattering paths and provides a three-dimensional confocal image of the sample as the latter one had become digitally transparent. The proof-of-concept experiment is performed on a human opaque cornea and an extension of the penetration depth by a factor five is demonstrated compared to the state-of-the-art.

The Proximal Tubule Response to Low Potassium Is Mediated by mTOR/AKT Activation via Kir4.2

Background: The renal epithelium is highly sensitive to changes in blood potassium (K+) levels. We have a detailed understanding of distal K+-sensitive signaling pathways, yet proximal mechanisms are less well-defined. Results: Mice lacking the basolateral proximal tubule inwardly rectifying K+ channel, Kir4.2, (Kir4.2−/−) demonstrated normal blood and urine electrolytes at baseline. Upon removal of K+ from their diet, Kir4.2−/− animals decompensated as evidenced by increased urinary K+ excretion and development of a proximal renal tubular acidosis compared to control animals (Kir4.2+/+). Potassium wasting in Kir4.2−/− mice was not proximal in origin, but was dependent upon increased ENaC activity along the connecting segment. This was demonstrated by the correction of K+ wasting in knockouts treated with the ENaC inhibitor, amiloride. Using lithium clearance studies, we further demonstrated increased distal sodium delivery in Kir4.2−/− animals due to reduced proximal tubule sodium reabsorption. Optical clearing and three-dimensional confocal imaging reveal Kir4.2−/− mice failed to undergo proximal tubule hypertrophy when fed a low K+ diet, while the distal convoluted tubule response was exaggerated in knockouts compared to control animals. A phosphoproteomics mass spectrometry analysis identified increased mTOR/AKT signaling as mediating the proximal dietary response to K+ depletion in control animals, and changes to mTOR/AKT signaling was blunted in Kir4.2 knockouts. Lastly, we demonstrated in isolated renal tubules that AKT phosphorylation in response to low K+ depended upon mTORC2 activation by secondary reductions in intracellular Cl−. Conclusions: In this work we identify the basolateral inwardly rectifying K+ channel, Kir4.2, as a mediator of the proximal tubule response to K+ deficiency. Kir4.2 knockout mice increase their distal nephron transport burden to maintain electrolyte homeostasis in the face of deficient proximal tubule cell function. Data identify a conserved proximal role for cell Cl− which, as it does along the distal convoluted tubule, responds to extracellular K+ changes to activate intracellular kinase signaling. Along the proximal tubule, mTOR/AKT signaling is essential for the cell response to low K+. Proximal and distal nephron cell Cl− responses work in concert to preserve K+ homeostasis. These studies were supported by NIH grants DP5-OD033412 (AST), DK51265, DK95785, DK62794, DK7569, P30DK114809 (RCH, MZZ), and the Vanderbilt Center for Kidney Disease and Vanderbilt Diabetes Research and Training Center (DK020593) pilot and feasibility grant (JSD). 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.

Electrophysiology and 3D-imaging reveal properties of human intracardiac neurons and increased excitability with atrial fibrillation.

Altered autonomic input to the heart plays a major role in atrial fibrillation (AF). Autonomic neurons termed ganglionated plexi (GP) are clustered on the heart surface to provide the last point of neural control of cardiac function. To date the properties of GP neurons in humans are unknown. Here we have addressed this knowledge gap in human GP neuron structure and physiology in patients with and without AF. Human right atrial GP neurons embedded in epicardial adipose tissue were excised during open heart surgery performed on both non-AF and AF patients and then characterised physiologically by whole cell patch clamp techniques. Structural analysis was also performed after fixation at both the single cell and at the entire GP levels via three-dimensional confocal imaging. Human GP neurons were found to exhibit unique properties and structural complexity with branched neurite outgrowth. Significant differences in excitability were revealed between AF and non-AF GP neurons as measured by lower current to induce action potential firing, a reduced occurrence of low action potential firing rates, decreased accommodation and increased synaptic density. Visualisation of entire GPs showed almost all neurons are cholinergic with a small proportion of noradrenergic and dual phenotype neurons. Phenotypic distribution differences occurred with AF including decreased cholinergic and dual phenotype neurons, and increased noradrenergic neurons. These data show both functional and structural differences occur between GP neurons from patients with and without AF, highlighting that cellular plasticity occurs in neural input to the heart that could alter autonomic influence on atrial function. KEY POINTS: The autonomic nervous system plays a critical role in regulating heart rhythm and the initiation of AF; however, the structural and functional properties of human autonomic neurons in the autonomic ganglionated plexi (GP) remain unknown. Here we perform the first whole cell patch clamp electrophysiological and large tissue confocal imaging analysis of these neurons from patients with and without AF. Our data show human GP neurons are functionally and structurally complex. Measurements of action potential kinetics show higher excitability in GP neurons from AF patients as measured by lower current to induce action potential firing, reduced low firing action potential rates, and decreased action potential accommodation. Confocal imaging shows increased synaptic density and noradrenergic phenotypes in patients with AF. Both functional and structural differences occur in GP neurons from patients with AF that could alter autonomic influence on atrial rhythm.

Elafin as a Prognostic Marker in Esophageal Squamous Cell Carcinoma: A Pilot Study Using Three-Dimensional Imaging and Genomic Profiling.

Esophageal cancers are globally the sixth deadliest malignancy, with limited curative options. The association of high serum elafin levels, a molecule produced by epithelial cells, with esophageal squamous cell carcinoma (ESCC) risk is established, but its link to poor ESCC prognosis remains unclear. To explore this question, we first used three-dimensional confocal imaging to create a model of the spatial distribution of elafin inside locoregional ESCC tissues. Then, after analyzing data obtained from whole-genome microarrays for ESCC cell lines and their more invasive sublines, we performed in vitro experiments using RNA sequencing to identify possible elafin-related pathways. Three-dimensional tissue imaging showed elafin distributed as an interweaved-like fibrous structure in the stroma of tissue obtained from patients with high serum levels of elafin and poorer prognoses. By contrast, the signal was confined inside or around the tumor nest in patients who had lower serum levels and better survival. The analysis of a TCGA dataset revealed that higher levels of elafin mRNA in stage I-IIIA ESCC patients were associated with shorter survival. The in vitro studies revealed that elafin promoted ESCC cell proliferation, migration, and invasion via the epithelial-mesenchymal transition pathway. Thus, elafin inhibition could potentially be used therapeutically to improve survival in patients with locoregional ESCC.

Quantitative Analysis of Differentiation Activity for Mouse Embryonic Stem Cells by Deep Learning for Cell Center Detection using Three-Dimensional Confocal Fluorescence Microscopy Images.

Accurate single cell segmentation provides means to monitor the behavior of single cell within a population of cells. Time-lapse fluorescence images are used to reveal heterogeneous nature of single mouse embryonic stem cell (ESC) colony and monitor fluctuations of the cell states. Automatic quantification of speed and status shifts of the ESCs depends on accurate single cell segmentation that is used to calculate the 3D center of every cell and track this cell for the quantification. This study proposes a new 3D U-net to accurately detect center of each single cell in 3D confocal images. The dimension of input 3D images to the U-net is flexible so that multiple center detections from different image directions can be implemented simultaneously to improve the center detection accuracy. This study showed that our method can improve accuracy for cell center detection and thus the quantification for ESC speeds and status shifts.

Mechanically manipulating glymphatic transport by ultrasound combined with microbubbles

The glymphatic system is a perivascular fluid transport system for waste clearance. Glymphatic transport is believed to be driven by the perivascular pumping effect created by the pulsation of the arterial wall caused by the cardiac cycle. Ultrasound sonication of circulating microbubbles (MBs) in the cerebral vasculature induces volumetric expansion and contraction of MBs that push and pull on the vessel wall to generate a MB pumping effect. The objective of this study was to evaluate whether glymphatic transport can be mechanically manipulated by focused ultrasound (FUS) sonication of MBs. The glymphatic pathway in intact mouse brains was studied using intranasal administration of fluorescently labeled albumin as fluid tracers, followed by FUS sonication at a deep brain target (thalamus) in the presence of intravenously injected MBs. Intracisternal magna injection, the conventional technique used in studying glymphatic transport, was employed to provide a comparative reference. Three-dimensional confocal microscopy imaging of optically cleared brain tissue revealed that FUS sonication enhanced the transport of fluorescently labeled albumin tracer in the perivascular space (PVS) along microvessels, primarily the arterioles. We also obtained evidence of FUS-enhanced penetration of the albumin tracer from the PVS into the interstitial space. This study revealed that ultrasound combined with circulating MBs could mechanically enhance glymphatic transport in the brain.

Intermediate-range order governs dynamics in dense colloidal liquids

The conventional wisdom is that liquids are completely disordered and lack nontrivial structure beyond nearest-neighbor distances. Recent observations have upended this view and demonstrated that the microstructure in liquids is surprisingly rich and plays a critical role in numerous physical, biological, and industrial processes. However, approaches to uncover this structure are either system-specific or yield results that are not physically intuitive. Here, through single-particle resolved three-dimensional confocal microscope imaging and the use of a recently introduced four-point correlation function, we show that bidisperse colloidal liquids have a highly nontrivial structure comprising alternating layers with icosahedral and dodecahedral order, which extends well beyond nearest-neighbor distances and grows with supercooling. By quantifying the dynamics of the system on the particle level, we establish that it is this intermediate-range order, and not the short-range order, which has a one-to-one correlation with dynamical heterogeneities, a property directly related to the relaxation dynamics of glassy liquids. Our experimental findings provide a direct and much sought-after link between the structure and dynamics of liquids and pave the way for probing the consequences of this intermediate-range order in other liquid state processes.

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.

New insights into the role of microheterogeneity of ZP3 during structural maturation of the avian equivalent of mammalian zona pellucida.

The egg coat including mammalian zona pellucida (ZP) and the avian equivalent, i.e., inner-perivitelline layer (IPVL), is a specialized extracellular matrix being composed of the ZP glycoproteins and surrounds both pre-ovulatory oocytes and ovulated egg cells in vertebrates. The egg coat is well known for its potential importance in both the reproduction and early development, although the underlying molecular mechanisms remain to be fully elucidated. Interestingly, ZP3, one of the ZP-glycoprotein family members forming scaffolds of the egg-coat matrices with other ZP glycoproteins, exhibits extreme but distinctive microheterogeneity to form a large number of isoelectric-point isoforms at least in the chicken IPVL. In the present study, we performed three-dimensional confocal imaging and two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE) of chicken IPVLs that were isolated from the ovarian follicles at different growth stages before ovulation. The results suggest that the relative proportions of the ZP3 isoforms are differentially altered during the structural maturation of the egg-coat matrices. Furthermore, tandem mass spectrometry (MS/MS) analyses and ZP1 binding assays against separated ZP3 isoforms demonstrated that each ZP3 isoform contains characteristic modifications, and there are large differences among ZP3 isoforms in the ZP1 binding affinities. These results suggest that the microheterogeneity of chicken ZP3 might be regulated to be associated with the formation of egg-coat matrices during the structural maturation of chicken IPVL. Our findings may provide new insights into molecular mechanisms of egg-coat assembly processes.

The photoreal new-age innovative pedagogical & counseling tool for glaucoma with 3D augmented reality (Eye MG AR).

In this manuscript, we have reported an augmented reality (AR) application named, 'Eye MG AR' innovated by us, to show different anatomical/pathological parts of the eyeball pertaining to glaucoma, from multiple customized angles of the user's choice to simplify glaucoma learning and clinical counseling. It is available free of cost from the Google Play Store for Android users. Procedures ranging from a simple outpatient department procedure (yttrium aluminium garnet peripheral iridotomy) to a complex surgical technique (trabeculectomy/tube surgery) can be explained and counseled with this Android application. Also, complex structures such as the angle of the anterior chamber and optic nerve head, are constructed in advanced real-time three-dimensional (3D) high-resolution confocal images. These 3D models are useful for glaucoma neophytes' immersive learning and 3D patient counseling experiences. This AR tool aims to reinvent the approach to glaucoma counseling with 'Unreal Engine' software and is created in a patient-friendly approach. Incepting 3D pedagogy and counseling with AR in glaucoma with real-time and high-resolution TrueColor confocal images has never been reported in the literature according to our knowledge.

Integrated multimodality microscope for accurate and efficient target-guided cryo-lamellae preparation

Cryo-electron tomography (cryo-ET) is a revolutionary technique for resolving the structure of subcellular organelles and macromolecular complexes in their cellular context. However, the application of the cryo-ET is hampered by the sample preparation step. Performing cryo-focused ion beam milling at an arbitrary position on the sample is inefficient, and the target of interest is not guaranteed to be preserved when thinning the cell from several micrometers to less than 300 nm thick. Here, we report a cryogenic correlated light, ion and electron microscopy (cryo-CLIEM) technique that is capable of preparing cryo-lamellae under the guidance of three-dimensional confocal imaging. Moreover, we demonstrate a workflow to preselect and preserve nanoscale target regions inside the finished cryo-lamellae. By successfully preparing cryo-lamellae that contain a single centriole or contact sites between subcellular organelles, we show that this approach is generally applicable, and shall help in innovating more applications of cryo-ET.

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.

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.

Highly Efficient Red/NIR-Emissive Fluorescent Probe with Polarity-Sensitive Character for Visualizing Cellular Lipid Droplets and Determining Their Polarity.

Lipid droplets (LDs), which are ubiquitous organelles existing in almost all eukaryotic cells, have attracted a lot of attention in the field of cell biology over the last decade. For the biological study of LDs via fluorescence imaging, the superior LD fluorescent probes with environmental polarity-sensitive character are highly desired and powerful but are very scarce. Herein, we have newly developed such a kind of fluorescent probe named LDs-Red which enables us to visualize LDs and to further reveal their polarity information. This fluorescent probe displays the advantages of intense red/near-infrared emission, high LD staining specificity, and good photostability; thus, it would be very useful for LD fluorescence imaging application. As a result, the three-dimensional confocal imaging to visualize spatial distribution of LDs and the multicolor confocal imaging to simultaneously observe LDs and other cellular organelles have been realized using this new LD fluorescent probe. Furthermore, the polarity-sensitive emission character of this probe enables us to quantitatively determine the LD polarity via spectral scan imaging. Consequently, the cancer cells (HepG2, HeLa, and Panc02) displaying lower polarity of LDs than the normal cells (L929, U251, and HT22) have been systematically demonstrated. In addition, this polarity-sensitive probe displaying shorter fluorescence wavelengths in cancer cells than in normal cells has an important and potential ability to distinguish them.

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids.

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Rapid Histological Assessment of Prostate Specimens in the Three-dimensional Space by Hydrophilic Tissue Clearing and Confocal Microscopy.

Microscopic examination of biopsied and resected prostatic specimens is the mainstay in the diagnosis of prostate cancer. However, conventional analysis of hematoxylin and eosin (H&E)-stained tissue is time-consuming and offers limited two-dimensional (2D) information. In the current study, we devised a method-termed Prostate Rapid Optical examination for cancer STATus (proSTAT)-for rapid screening of prostate cancer using high-resolution 2D and three-dimensional (3D) confocal images obtained after hydrophilic tissue clearing of 100-µm-thick tissue slices. The results of the proSTAT method were compared with those of traditional H&E stains for the analysis of cores (n=15) obtained from radical prostatectomy specimens (n=5). Gland lumen formation, consistent with Gleason pattern 3, was evident following tracking of multiple optical imaging sections. In addition, 3D rendering allowed visualizing a tubular network of interconnecting branches. Rapid 3D fluorescent labeling of tumor protein p63 accurately distinguished prostate adenocarcinoma from normal tissue and benign lesions. Compared with conventional stains, the 3D spatial and molecular information extracted from proSTAT may significantly increase the amount of available data for pathological assessment of prostate specimens. Our approach is amenable to automation and-subject to independent validation-can find a wide spectrum of clinical and research applications.

Early transverse tubule involvement in cardiomyocytes in hereditary transthyretin amyloidosis: a possible cause of cardiac events

BackgroundCardiac involvement is one of the most frequent and fatal manifestations of hereditary transthyretin (ATTRv) amyloidosis. This study sought to clarify the pathogenesis of ATTRv amyloidosis, specifically, how transthyretin (TTR) amyloid begins to deposit in cardiomyocytes and how this deposition progresses in these cells. MethodsWe analyzed autopsy cardiac tissues from five patients with ATTRv amyloidosis by using confocal microscopy with thioflavin S staining and immunofluorescence and electron microscopy to demonstrate the pattern of TTR amyloid deposition in cardiomyocytes. ResultsWe demonstrated predominant amyloid deposition in the transverse tubules (t-tubules) of cardiomyocytes at the early stage of TTR amyloid deposition. Also, a pattern of the progression of amyloid deposition from deeply invaginated extracellular matrix, that is, t-tubules, to cell surface extracellular matrix, that is, basement membrane, was noted. Three-dimensional confocal microscopic images revealed the abnormal architecture of the t-tubules with nodular swelling, branching, and confluence in the cardiomyocytes with amyloid deposition. Double immunofluorescence staining with anti-TTR antibody and CACNA1C antibody demonstrated reduced voltage-dependent calcium channels around amyloid deposition. ConclusionsOur pathological study demonstrated that t-tubule involvement is an early event in cardiomyocytes in the pathogenesis of ATTRv amyloidosis. This finding may indicate that disruption of t-tubules in cardiomyocytes may contribute to the pathogenesis of cardiac events including heart failure and arrhythmia.

U-net structures for segmentation of single mouse embryonic stem cells using three-dimensional confocal microscopy images.

Cell segmentation at a single cell resolution is required to provide insights for basic biology and application study. However, there are issues of low signal-to-noise ratio, weak fluorescence response, and insufficient resolution along the image stacking direction in 3D confocal images (volume). It has been difficult to segment out single cells from close or contacted cells in a cell volume using image processing methods or together with geometric processing methods. Recently, 3D deep learning methods have been used to avoid tedious parameter settings in the image and geometric processing, but still not easy to segment out close or contacted single cells. This paper proposes a 2D U-net to segment cell regions in high accuracy and computing performance. Better 3D cell images and single cell segmentation for close or contacted cells are achieved by combining a 3D U-net to detect the centers of single cells in the volume.

Scaffold Pore Curvature Influences ΜSC Fate through Differential Cellular Organization and YAP/TAZ Activity.

Tissue engineering aims to repair, restore, and/or replace tissues in the human body as an alternative to grafts and prostheses. Biomaterial scaffolds can be utilized to provide a three-dimensional microenvironment to facilitate tissue regeneration. Previously, we reported that scaffold pore size influences vascularization and extracellular matrix composition both in vivo and in vitro, to ultimately influence tissue phenotype for regenerating cranial suture and bone tissues, which have markedly different tissue properties despite similar multipotent stem cell populations. To rationally design biomaterials for specific cell and tissue fate specification, it is critical to understand the molecular processes governed by cell-biomaterial interactions, which guide cell fate specification. Building on our previous work, in this report we investigated the hypothesis that scaffold pore curvature, the direct consequence of pore size, modulates the differentiation trajectory of mesenchymal stem cells (MSCs) through alterations in the cytoskeleton. First, we demonstrated that sufficiently small pores facilitate cell clustering in subcutaneous explants cultured in vivo, which we previously reported to demonstrate stem tissue phenotype both in vivo and in vitro. Based on this observation, we cultured cell-scaffold constructs in vitro to assess early time point interactions between cells and the matrix as a function of pore size. We demonstrate that principle curvature directly influences nuclear aspect and cell aggregation in vitro. Scaffold pores with a sufficiently low degree of principle curvature enables cell differentiation; pharmacologic inhibition of actin cytoskeleton polymerization in these scaffolds decreased differentiation, indicating a critical role of the cytoskeleton in transducing cues from the scaffold pore microenvironment to the cell nucleus. We fabricated a macropore model, which allows for three-dimensional confocal imaging and demonstrates that a higher principle curvature facilitates cell aggregation and the formation of a potentially protective niche within scaffold macropores which prevents MSC differentiation and retains their stemness. Sufficiently high principle curvature upregulates yes-associated protein (YAP) phosphorylation while decreased principle curvature downregulates YAP phosphorylation and increases YAP nuclear translocation with subsequent transcriptional activation towards an osteogenic differentiation fate. Finally, we demonstrate that the inhibition of the YAP/TAZ pathway causes a defect in differentiation, while YAP/TAZ activation causes premature differentiation in a curvature-dependent way when modulated by verteporfin (VP) and 1-oleyl-lysophosphatidic acid (LPA), respectively, confirming the critical role of biomaterials-mediated YAP/TAZ signaling in cell differentiation and fate specification. Our data support that the principle curvature of scaffold macropores is a critical design criterion which guides the differentiation trajectory of mesenchymal stem cells’ scaffolds. Biomaterial-mediated regulation of YAP/TAZ may significantly contribute to influencing the regenerative outcomes of biomaterials-based tissue engineering strategies through their specific pore design.