Three-dimensional Confocal Microscopy Research Articles

Enhanced wastewater treatment by modified basalt fiber bio-carriers: Effect of etching and surface functionalization

The application of biological carriers holds a central position in wastewater treatment for biofilm-mediated degradation processes. In this study, the surface of basalt fibers (BF) was modified by combined etching and grouping (surface functionalization via attaching glutamic acid) to produce MBFEG. The etching modification enhanced the surface properties and specific surface area of BF. However, combined etching and functionalization resulted in the successful loading of amino acids on the surface of MBFEG, ultimately improving surface hydrophilicity, adsorption properties, and biophilicity of the bio-carrier. Three-dimensional confocal laser scanning microscopy confirmed that the combined modification was highly suitable in improving microbial activity and biophilicity. The microbial communities on MBFEG bio-carrier were rich and diverse indicating a stronger degree of biogeochemical cycling, indicating enrichment of nitrogen and carbon cycles. Conclusively, etching and grouping modification technology hold a promising future for BF application in biological wastewater treatment schemes.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis.

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Biodegradable PCL-b-PLA Microspheres with Nanopores Prepared via RAFT Polymerization and UV Photodegradation of Poly(Methyl Vinyl Ketone) Blocks.

Biodegradable triblock copolymers based on poly(ε-caprolactone) (PCL) and poly(lactic acid) (PLA) were synthesized via ring-opening polymerization of L-lactide followed by reversible addition–fragmentation chain-transfer (RAFT) polymerization of poly(methyl vinyl ketone) (PMVK) as a photodegradable block, and characterized by FT-IR and 1H NMR spectroscopy for structural analyses, and by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) for their thermal properties. Porous, biodegradable PCL-b-PLA microspheres were fabricated via the oil/water (O/W) emulsion evaporation method, followed by photodegradation of PMVK blocks by UV irradiation. The macro-chain transfer agent (CTA) synthesized by reacting a carboxylic-acid-terminated CTA—S-1-dodecyl-S′-(a,a′-dimethyl-a′′-acetic acid)trithiocarbonate (DDMAT)—with a hydroxyl-terminated PCL-b-PLA block copolymer was used to synthesize well-defined triblock copolymers with methyl vinyl ketone via RAFT polymerization with controlled molecular weights and narrow polydispersity. Gel permeation chromatography traces indicated that the molecular weight of the triblock copolymer decreased with UV irradiation time because of the photodegradation of the PMVK blocks. The morphology of the microspheres before and after UV irradiation was investigated using SEM and videos of three-dimensional confocal laser microscopy, showing a change in their surface texture from smooth to rough, with high porosity owing to the photodegradation of the PMVK blocks to become porous templates.

Ductal Macrophages Predominate in the Immune Landscape of the Lactating Mammary Gland.

The mammary gland is unique in female mammals. Mammary tissue undergoes development and remodeling during lactation, a stage associated with high susceptibility to bacterial infections, inducing an inflammatory condition called mastitis. Although the immune response of the mammary gland has been the subject of intense research to improve prevention and treatment efficacy, the precise definition of its immune composition at this particular physiological stage is still missing. We combined single-cell RNA-Seq, flow cytometry, and three-dimensional confocal microscopy techniques to characterize the immune landscape of lactating murine mammary tissue. Macrophages dominated the immune cell repertoire and could be subdivided into at least two subsets: ductal and stromal macrophages. Ductal macrophages represented approximately 80% of the total CD45pos immune cells and co-expressed F4/80 and CD11c, with high levels of MHC class II molecules. They were strategically poised below the alveolar basal cells in contact with the myoepithelial cell network. Adaptive T and B lymphocytes were remarkably less numerous at this stage, which could explain the limited efficacy of vaccination against mastitis. These results support the view that new strategies to increase mammary immunity and prevent mastitis should be devised.

Effect of La2Sn2O7 content on Ag-La2Sn2O7/SnO2 arc erosion behavior and mechanism

La2Sn2O7/SnO2 powder was synthesized by chemical co-precipitation method, and Ag-La2Sn2O7/SnO2 composites were prepared by hot-pressing sintering. The electrical resistivity, density, Brinell hardness and flexural strength of Ag-La2Sn2O7/SnO2 composites were measured. Moreover, the effect of La2Sn2O7 content on the arc erosion behavior of Ag-La2Sn2O7/SnO2 composites at 7 kV voltage was studied. With the increase of La2Sn2O7 content, the arc duration and arc energy decrease, and the breakdown strength increases. The surface morphology of Ag-La2Sn2O7/SnO2 composites after arc erosion was investigated using scanning electron microscopy and three-dimensional laser confocal scanning microscopy. With the increase of La2Sn2O7 content, the smaller the erosion damage, the better the anti-arc erosion performance. However, too much La2Sn2O7 results in the decrease of Ag-La2Sn2O7/SnO2 properties and severe arc erosion. X-ray photoelectron spectroscopy was used to determine the composition of the erosion region. The effect of La2Sn2O7 on wettability between Ag and SnO2 was investigated, and the erosion mechanism of Ag-La2Sn2O7/SnO2 composites was discussed systematically. This study can provide a reference for the application of Ag matrix electrical contact materials in high-voltage electrical appliances.

Overloading stress-induced progressive degeneration and self-repair in condylar cartilage.

Overloading stress-induced condylar cartilage degeneration acts as the main pathologic change in temporomandibular joint osteoarthritis (TMJ-OA). However, the progression of degeneration and the ability for self-repair remain poorly understood. Here, we explored the progression of cartilage degeneration by dividing pathological stages using a steady mouth-opening mouse model. Then, we observed changes of cartilage by removing the loading at different stages to test the potential self-repair after degeneration induced. Three-dimensional confocal microscopy combined with histology and micro-CT scanning was applied to examine TMJ at different stages of degeneration before and after self-repair. We found the cartilage underwent progressive and thorough degeneration as the overloading stress developed. During the initial adaptation stage, robust proliferation of posteromedial cartilage began at the area of direct loading. Subsequently, widespread chondrocyte apoptosis was found, followed by new chondrocyte proliferation in aggregates with matrix degradation and subchondral bone catabolism. Finally, with cartilage surface damage, the degeneration reached a point where the lesion could not be reversed by self-repair. While the cartilage nearly returned to normal when the interference was removed within 5 days. These results suggested overloading force induces a pathological process of successive degeneration in TMJ cartilage, which can be reversed by self-repair at early stages.

Chemical Imaging of Single Anisotropic Polystyrene/Poly (Methacrylate) Microspheres with Complex Hierarchical Architecture.

Monodisperse polystyrene spheres are functional materials with interesting properties, such as high cohesion strength, strong adsorptivity, and surface reactivity. They have shown a high application value in biomedicine, information engineering, chromatographic fillers, supercapacitor electrode materials, and other fields. To fully understand and tailor particle synthesis, the methods for characterization of their complex 3D morphological features need to be further explored. Here we present a chemical imaging study based on three-dimensional confocal Raman microscopy (3D-CRM), scanning electron microscopy (SEM), focused ion beam (FIB), diffuse reflectance infrared Fourier transform (DRIFT), and nuclear magnetic resonance (NMR) spectroscopy for individual porous swollen polystyrene/poly (glycidyl methacrylate-co-ethylene di-methacrylate) particles. Polystyrene particles were synthesized with different co-existing chemical entities, which could be identified and assigned to distinct regions of the same particle. The porosity was studied by a combination of SEM and FIB. Images of milled particles indicated a comparable porosity on the surface and in the bulk. The combination of standard analytical techniques such as DRIFT and NMR spectroscopies yielded new insights into the inner structure and chemical composition of these particles. This knowledge supports the further development of particle synthesis and the design of new strategies to prepare particles with complex hierarchical architectures.

Plant-based oral care product exhibits antibacterial effects on different stages of oral multispecies biofilm development in vitro

BackgroundExcessive biofilm formation on surfaces in the oral cavity is amongst the main reasons for severe infection development like periodontitis and peri-implantitis. Mechanical biofilm removal as well as the use of adjuvant antiseptics supports the prevention of pathogenic biofilm formation. Recently, the antibacterial effect of the oral care product REPHA-OS®, based on medicinal plant extracts and essential oils, has been demonstrated on oral pathogens grown on agar plates. In the present study, the effectiveness of the product on medical relevant oral biofilm development should be demonstrated for the first time.MethodsAn established in vitro oral multispecies biofilm, composed of Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis, was used to analyze the antibacterial effect of different REPHA-OS® concentrations on planktonic bacteria, biofilm formation and mature biofilms. It was quantified using metabolic activity assays and live/dead fluorescence staining combined with three-dimensional confocal laser-scanning microscopy. Additionally, effects on species distribution inside the biofilm were assessed by means of quantitative real-time PCR.ResultsREPHA-OS® showed statistically significant antimicrobial effects on all stages of biofilm development: a minimal inhibitory concentration of 5% could be detected for both, for planktonic bacteria and for biofilm formation. Interestingly, only a slightly higher concentration of 10% was necessary to completely kill all bacteria in mature biofilms also. In contrast, an influence on the biofilm matrix or the species distribution could not be observed. The effect could be attributed to the herbal ingredients, not to the contained ethanol.ConclusionThe strong antibacterial effect of REPHA-OS® on different stages of oral biofilm development strengthens its application as an alternative adjuvant in oral care therapies.

Mesoscopic protein-rich clusters host the nucleation of mutant p53 amyloid fibrils

The protein p53 is a crucial tumor suppressor, often called "the guardian of the genome"; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.

Anomalous mechanics of Zn2+-modified fibrin networks

Fibrin is the main component of blood clots. The mechanical properties of fibrin are therefore of critical importance in successful hemostasis. One of the divalent cations released by platelets during hemostasis is Zn2+; however, its effect on the network structure of fibrin gels and on the resultant mechanical properties remains poorly understood. Here, by combining mechanical measurements with three-dimensional confocal microscopy imaging, we show that Zn2+ can tune the fibrin network structure and alter its mechanical properties. In the presence of Zn2+, fibrin protofibrils form large bundles that cause a coarsening of the fibrin network due to an increase in fiber diameter and reduction of the total fiber length. We further show that the protofibrils in these bundles are loosely coupled to one another, which results in a decrease of the elastic modulus with increasing Zn2+ concentrations. We explore the elastic properties of these networks at both low and high stress: At low stress, the elasticity originates from pulling the thermal slack out of the network, and this is consistent with the thermal bending of the fibers. By contrast, at high stress, the elasticity exhibits a common master curve consistent with the stretching of individual protofibrils. These results show that the mechanics of a fibrin network are closely correlated with its microscopic structure and inform our understanding of the structure and physical mechanisms leading to defective or excessive clot stiffness.

Process parameters effect on high-temperature friction and galling characteristics of AA7075 sheets

ABSTRACT During hot forming of high-strength 7xxx series alloy, friction and galling often occur on the contact surface, degrade product surfaces quality, and affect severely by the process parameters, especially by load and speed. In the present paper, the effect of process parameters, namely applied load and sliding speed, on high-temperature friction and galling performance of AA7075 sheets has been determined using linear sliding tests at 350°C in simulated hot forming. The microscopic observations, i.e., scanning electron microscopy (SEM) and three-dimensional confocal scanning optical microscopy, were utilized to analyze hard phase distribution of the alloy as well as surface and sub-surface of the posttest specimens. A friction model was proposed to describe the friction mechanism. The results show that wear rate gradually decreased with increasing load and speed, attributing to the formed compacted oxide layer on the worn surface, which inhibited the rate of further attrition. Besides, for all test conditions, adhesion dominated the wear mechanism, and the compacted layer was damaged more serious due to more initiation of micro-cracks between hard phase and matrix and higher contact interface temperature at higher load and speed, thus the extent of adhesion is grievous resulting in a higher wear loss.

Intracellular bacteria are common and taxonomically diverse in cultured and in hospite algal endosymbionts of coral reefs

Corals house a variety of microorganisms which they depend on for their survival, including endosymbiotic dinoflagellates (Symbiodiniaceae) and bacteria. While cnidarian–microorganism interactions are widely studied, Symbiodiniaceae–bacteria interactions are only just beginning to receive attention. Here, we describe the localization and composition of the bacterial communities associated with cultures of 11 Symbiodiniaceae strains from nine species and six genera. Three-dimensional confocal laser scanning and electron microscopy revealed bacteria are present inside the Symbiodiniaceae cells as well as closely associated with their external cell surface. Bacterial pure cultures and 16S rRNA gene metabarcoding from Symbiodiniaceae cultures highlighted distinct and highly diverse bacterial communities occur intracellularly, closely associated with the Symbiodiniaceae outer cell surface and loosely associated (i.e., in the surrounding culture media). The intracellular bacteria are highly conserved across Symbiodiniaceae species, suggesting they may be involved in Symbiodiniaceae physiology. Our findings provide unique new insights into the biology of Symbiodiniaceae.

Regulating the Nb2C nanosheets with different degrees of oxidation in water lubricated sliding toward an excellent tribological performance

Novel two-dimensional (2D) Nb2C nanosheets were successfully prepared through a simple lultrasonic and magnetic stirring treatment from the original accordion-like powder. To further study their water-lubrication properties and deal with common oxidation problems, Nb2C nanosheets with different oxidation degrees were prepared and achieved long-term stability in deionized water. Scanning electron microscope (SEM), transmission electron microscope (TEM), scanning probe microscope (SPM), X-ray powder diffraction (XRD), Raman, and X-ray photoelectron spectrometer (XPS) experiments were utilized to characterize the structure, morphology, and dispersion of Nb2C nanosheets with different degrees of oxidation. The tribological behaviors of Nb2C with different degrees of oxidation as additives for water lubrication were characterized using a UMT-3 friction testing machine. The wear scars formed on the 316 steel surface were measured using three-dimensional (3D) laser scanning confocal microscopy. The tribological results showed that a moderately oxidized Nb2C nanosheet, which owned the composition of Nb2C/Nb2O5/C, displayed excellent tribological performance, with the friction coefficient (COF) decreasing by 90.3% and a decrease in the wear rate by 73.1% compared with pure water. Combining the TEM and Raman spectra, it was shown that Nb2O5 nanoparticles filled in the worn zone, and the layered Nb2C and C were adsorbed into the surface of the friction pair to form a protective lubricating film. This combined action resulted in an excellent lubricating performance.

Neuroglia infection by rabies virus after anterograde virus spread in peripheral neurons

The highly neurotropic rabies virus (RABV) enters peripheral neurons at axon termini and requires long distance axonal transport and trans-synaptic spread between neurons for the infection of the central nervous system (CNS). Recent 3D imaging of field RABV-infected brains revealed a remarkably high proportion of infected astroglia, indicating that highly virulent field viruses are able to suppress astrocyte-mediated innate immune responses and virus elimination pathways. While fundamental for CNS invasion, in vivo field RABV spread and tropism in peripheral tissues is understudied. Here, we used three-dimensional light sheet and confocal laser scanning microscopy to investigate the in vivo distribution patterns of a field RABV clone in cleared high-volume tissue samples after infection via a natural (intramuscular; hind leg) and an artificial (intracranial) inoculation route. Immunostaining of virus and host markers provided a comprehensive overview of RABV infection in the CNS and peripheral nerves after centripetal and centrifugal virus spread. Importantly, we identified non-neuronal, axon-ensheathing neuroglia (Schwann cells, SCs) in peripheral nerves of the hind leg and facial regions as a target cell population of field RABV. This suggests that virus release from axons and infected SCs is part of the RABV in vivo cycle and may affect RABV-related demyelination of peripheral neurons and local innate immune responses. Detection of RABV in axon-surrounding myelinating SCs after i.c. infection further provided evidence for anterograde spread of RABV, highlighting that RABV axonal transport and spread of infectious virus in peripheral nerves is not exclusively retrograde. Our data support a new model in which, comparable to CNS neuroglia, SC infection in peripheral nerves suppresses glia-mediated innate immunity and delays antiviral host responses required for successful transport from the peripheral infection sites to the brain.

High-voltage arc erosion behavior and mechanism of Ti3AlC2 under different ambient atmospheres

The arc erosion behavior of Ti3AlC2 in oxygen, air, nitrogen, carbon dioxide, argon, and sulfur hexafluoride atmospheres at 9 kV voltage was studied. The breakdown strength increased in the order of oxygen, air, carbon dioxide, nitrogen, argon, and sulfur hexafluoride, whereas the arc energy decreased. Cracks, pits, and bulges on the eroded surface were investigated using scanning electron microscopy and three-dimensional (3D) laser confocal scanning microscopy. Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to determine the composition of the eroded surface. The arc energy and electromagnetic force lead to the formation of erosion characteristics. The mechanism of erosion under the different atmospheres is discussed systematically, and is called the “decomposition-oxidation” process in oxygen, air, and carbon dioxide and the “decomposition-rereaction” process in nitrogen and sulfur hexafluoride. This study provides a reference for the application of MAX phase materials in high-voltage electrical appliances.

DMD-based three-dimensional chromatic confocal microscopy.

In this paper, a digital mirror device (DMD)-based chromatic confocal microscopy is proposed and demonstrated for three-dimensional (3D) surface profiling without any mechanical scanning. In this method, the DMD works as the multipoint source and multi-pinhole at the same time to achieve the lateral scanning. Moreover, axial scanning is realized through the chromatic aberration of the confocal optics. Since the micromirror array of the DMD is not perpendicular to the confocal imaging axis, a corresponding calibration is needed to eliminate the tilt effects and perform accurate 3D imaging. The measurement range with the current optical system is 45 µm over 505-650 nm working spectrum and can be increased by using a custom objective with large chromatic aberration. The system performance has been demonstrated with a multistep sample.

Non-Proliferative Diabetic Retinopathy Is Characterized by Nonuniform Alterations of Peripapillary Capillary Networks.

PurposeThe purpose of this study was to use three-dimensional confocal microscopy to quantify the spatial patterns of capillary network alterations in nonproliferative diabetic retinopathy (NPDR).MethodsThe retinal microvasculature was perfusion-labelled in seven normal human donor eyes and six age-matched donor eyes with NPDR. The peripapillary microcirculation was studied using confocal scanning laser microscopy. Capillary density and diameters of the radial peripapillary capillary plexus (RPCP), superficial capillary plexus (SCP), intermediate capillary plexus (ICP), and deep capillary plexus (DCP) were quantified and compared. Three-dimensional visualization strategies were also used to compare the communications between capillary beds and precapillary arterioles and postcapillary venules.ResultsMean capillary diameter was significantly increased in the NPDR group (P < 0.001). Intercapillary distance was significantly increased in the DCP (P = 0.004) and RPCP (P = 0.022) of the NPDR group (P = 0.010) but not the SCP (P = 0.155) or ICP (P = 0.103). The NPDR group was associated with an increased frequency of inflow communication between the SCP and ICP/DCP and a decreased frequency of communication between the SCP and RPCP (P = 0.023). There was no difference in the patterns of outflow communications between the two groups (P = 0.771).ConclusionsThis study demonstrates that capillary plexuses are nonuniformly perturbed in NPDR. These structural changes may be indicative of perturbations to blood flow patterns between different retinal layers. Our findings may aid the interpretation of previous clinical observations made using optical coherence tomography angiography as well as improve our understanding of the pathogenesis of NPDR.

Width determination for deep grooves based on a variable point spread function imaging model.

In three-dimensional confocal microscopy, two-dimensional width measurement can be significantly influenced by the groove height. The groove height not only results in deformation of the input light field due to the effect of edge occlusions, but also introduces a defocus error to the detection plane. This paper proposes a new, to the best of our knowledge, edge-setting method to determine groove width, which engineers the point spread function to correct for the groove edge obstruction effect and develops an edge obstruction imaging model (EOIM) based on the variable point spread function. This model gives a relationship between the groove height and the normalized intensity at the groove edge and can use this relationship to determine the groove edge position that would result from focusing at the groove's lower surface. Experimental results show that an EOIM-based width determination method is more accurate than the traditional 1/4 edge-setting method. Compared to the 1/4 edge-setting method, the deviation from a reference width measured with traceable scanning electron microscopy is reduced by a factor of 2.1 with a 1.3 times smaller standard deviation.

Murine Intraepithelial Dendritic Cells Interact With Phagocytic Cells During Aspergillus fumigatus-Induced Inflammation.

People are constantly exposed to airborne fungal spores, including Aspergillus fumigatus conidia that can cause life-threatening conditions in immunocompromised patients or acute exacerbations in allergics. However, immunocompetent hosts do not exhibit mycoses or systemic inflammation, due to the sufficient but not excessive antifungal immune response that prevent fungal invasion. Intraepithelial dendritic cells (IE-DCs) of the conducting airway mucosa are located in the primary site of the inhalant pathogen entry; these cells can sense A. fumigatus conidia and maintain homeostasis. The mechanisms by which IE-DCs contribute to regulating the antifungal immune response and controlling conidia dissemination are not understood. To clarify the role of IE-DCs in the balance between pathogen sensing and immune tolerance we investigated the A. fumigatus conidia distribution in optically cleared mouse lungs and estimated the kinetics of the local phagocytic response during the course of inflammation. MHCII+ antigen-presenting cells, including IE-DCs, and CD11b+ phagocytes were identified by immunohistochemistry and three-dimensional fluorescence confocal laser-scanning microscopy of conducting airway whole-mounts. Application of A. fumigatus conidia increased the number of CD11b+ phagocytes in the conducting airway mucosa and induced the trafficking of these cells through the conducting airway wall to the luminal side of the epithelium. Some CD11b+ phagocytes internalized conidia in the conducting airway lumen. During the migration through the airway wall, CD11b+ phagocytes formed clusters. Permanently located in the airway wall IE-DCs contacted both single CD11b+ phagocytes and clusters. Based on the spatiotemporal characteristics of the interactions between IE-DCs and CD11b+ phagocytes, we provide a novel anatomical rationale for the contribution of IE-DCs to controlling the excessive phagocyte-mediated immune response rather than participating in pathogen uptake.

Location and function of transient receptor potential canonical channel 1 in ventricular myocytes

Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca2+ signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca2+ ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca2+ signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na+ and Ca2+ free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca2+ content, respectively. In isolated rabbit cardiomyocytes bathed in Na+ and Ca2+ free solution, we found an increased decay of the cytosolic Ca2+ concentration [Ca2+]i and increased SR Ca2+ content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca2+ leak through SR TRPC channels increased the systolic and diastolic [Ca2+]i with only minor effects on the action potential and SR Ca2+ content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca2+ leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will guide us to an understanding of TRPC1 channels as modulators of [Ca2+]i and contractility in cardiomyocytes.