A deep learning approach to identifying immunogold particles in electron microscopy images.

Electron microscopy (EM) enables high-resolution visualization of protein distributions in biological tissues. For detection, gold nanoparticles are typically used as an electron-dense marker for immunohistochemically labeled proteins. Manual annotation of gold particle labels is laborious and time consuming, as gold particle counts can exceed 100,000 across hundreds of image segments to obtain conclusive data sets. To automate this process, we developed Gold Digger, a software tool that uses a modified pix2pix deep learning network capable of detecting and annotating colloidal gold particles in biological EM images obtained from both freeze-fracture replicas and plastic sections prepared with the post-embedding method. Gold Digger performs at near-human-level accuracy, can handle large images, and includes a user-friendly tool with a graphical interface for proof reading outputs by users. Manual error correction also helps for continued re-training of the network to improve annotation accuracy over time. Gold Digger thus enables rapid high-throughput analysis of immunogold-labeled EM data and is freely available to the research community.

Biodistribution of colloidal gold nanoparticles after intravenous injection: Effects of PEGylation at the same particle size.

BACKGROUND: Recently, polyethylene glycol (PEG) modified gold nanoparticles have been studied to maintaining long-term stability in biological fluids. Its biodistribution was also reported, however, comparison of bare gold nanoparticles and PEGylated gold nanoparticles with equal particle size is not sufficient. OBJECTIVE: We prepared bare gold nanoparticles and PEGylated gold nanoparticles with diameters of 20-30-nm or 50-nm to avoid the influence of particle diameter, and studied their biodistribution in the mouse. METHODS: Gold concentrations in brain, heart, lungs, liver, stomach, pancreas, spleen, kidneys, blood, urine, and feces were measured at 0.5, 1, 2, 3, 6, 12, 18, 24, and 48 h after administration of gold nanoparticles using inductively coupled plasma atomic emission spectrometry. RESULTS: At 48 h after intravenous administration, accumulation in the liver and spleen was significantly reduced by PEGylation, and the gold amounts of PEGylated gold nanoparticles with diameters of 20-30 nm and 50-nm in the brain were 3.6 times and 2.7 times higher than those of bare gold nanoparticles, respectively. CONCLUSIONS: These results indicated that the usefulness of PEGylated gold nanoparticles with small particle size for a drug carrier.

A theranostic dental pulp capping agent with improved MRI and CT contrast and biological properties.

Different materials have been used for vital dental pulp treatment. Preferably a pulp capping agent should show appropriate biological performance, excellent handling properties, and a good imaging contrast. These features can be delivered into a single material through the combination of therapeutic and diagnostic agents (i.e. theranostic). Calcium phosphate based composites (CPCs) are potentially ideal candidate for pulp treatment, although poor imaging contrast and poor dentino-inductive properties are limiting their clinical use. In this study, a theranostic dental pulp capping agent was developed. First, imaging properties of the CPC were improved by using a core-shell structured dual contrast agent (csDCA) consisting of superparamagnetic iron oxide (SPIO) and colloidal gold, as MRI and CT contrast agent respectively. Second, biological properties were implemented by using a dentinogenic factor (i.e. bone morphogenetic protein 2, BMP-2). The obtained CPC/csDCA/BMP-2 composite was tested in vivo, as direct pulp capping agent, in a male Habsi goat incisor model. Our outcomes showed no relevant alteration of the handling and mechanical properties (e.g. setting time, injectability, and compressive strength) by the incorporation of csDCA particles. In vivo results proved MRI contrast enhancement up to 7weeks. Incisors treated with BMP-2 showed improved tertiary dentin deposition as well as faster cement degradation as measured by µCT assessment. In conclusion, the presented theranostic agent matches the imaging and regenerative requirements for pulp capping applications. STATEMENT OF SIGNIFICANCE: In this study, we combined diagnostic and therapeutic agents in order to developed a theranostic pulp capping agent with enhanced MRI and CT contrast and improved dentin regeneration ability. In our study we cover all the steps from material preparation, mechanical and in vitro characterization, to in vivo study in a goat dental model. To the best of our knowledge, this is the first time that a theranostic pulp capping material have been developed and tested in an in vivo animal model. Our promising results in term of imaging contrast enhancement and of induction of new dentin formation, open a new scenario in the development of innovative dental materials.

Biodistribution and excretion of colloidal gold nanoparticles after intravenous injection: Effects of particle size.

BACKGROUND: Inorganic gold nanoparticles (NPs) have a huge potential in targeted drug delivery. Simple preparation and surface modification procedure with their special physicochemical properties of gold NPs attract their use such as tumor targeting and the detection of cancerous cell. OBJECTIVE: Various studies were reported, however, details of biodistribution profile of gold NPs were not enough evaluated. We have studied biodistribution profile of gold NPs having various particle sizes (20, 50 and 100 nm). METHODS: Gold concentrations in brain, heart, lungs, liver, stomach, pancreas, spleen, kidneys, blood, urine, and feces were measured at 5 minutes, 0.25, 0.5, 1, 2, 3, 6, 12, 18 and 24 hours after administration of gold NPs using inductively coupled plasma atomic emission spectrometry. RESULTS: In lungs and brain, especially 20-nm gold NPs were accumulated after 2-3 hours of dose administration, and they were kept for 24 hours, whereas they showed relatively low accumulation in heart, stomach and pancreas. After 12 hours, 3.3-14.4% of the injected gold were observed in fecal matter and urine. CONCLUSIONS: From this study, the application of gold NPs for targeted delivery to lungs and brain and the excretion route of the gold NPs from the body were suggested.

DNA-gadolinium-gold nanoparticles for in vivo T1 MR imaging of transplanted human neural stem cells.

The unambiguous imaging of transplanted cells remains a major challenge to understand their biological function and therapeutic efficacy. In vivo imaging of implanted cells is reliant on tagging these to differentiate them from host tissue, such as the brain. We here characterize a gold nanoparticle conjugate that is functionalized with modified deoxythymidine oligonucleotides bearing Gd(III) chelates and a red fluorescent Cy3 moiety to visualize in vivo transplanted human neural stem cells. This DNA-Gd@Au nanoparticle (DNA-Gd@AuNP) exhibits an improved T1 relaxivity and excellent cell uptake. No significant effects of cell uptake have been found on essential cell functions. Although T1 relaxivity is attenuated within cells, it is sufficiently preserved to afford the in vivo detection of transplanted cells using an optimized voxel size. In vivo MR images were corroborated by a post-mortem histological verification of DNA-Gd@AuNPs in transplanted cells. With 70% of cells being correctly identified using the DNA-Gd-AuNPs indicates an overall reliable detection. Less than 1% of cells were false positive for DNA-Gd@AuNPs, but a significant number of 30% false negatives reveals a dramatic underestimation of transplanted cells using this approach. DNA-Gd@AuNPs therefore offer new opportunities to visualize transplanted cells unequivocally using T1 contrast and use cellular MRI as a tool to derive biologically relevant information that allows us to understand how the survival and location of implanted cells determines therapeutic efficacy.

Injectable colloidal gold in a sucrose acetate isobutyrate gelating matrix with potential use in radiation therapy.

External beam radiation therapy relies on the ability to deliver high radiation doses to tumor cells with minimal exposure to surrounding healthy tissue. Advanced irradiation techniques, including image-guided radiation therapy (IGRT), rely on the ability to locate tumors to optimize the therapeutic benefit of these techniques. Today, radiopaque fiducial tissue markers are placed in or around tumors, for example, in prostate cancer patients to enhance the precision of daily and/or real-time IGRT. A liquid injectable fiducial marker (nanogel) is developed based on PEGylated gold nanoparticles and sucrose acetate isobutyrate (SAIB) with improved properties compared to current solid fiducial markers. The developed nanogel is investigated in vitro and subsequently evaluated in vivo in immunocompetent NMRI mice. The nanogel shows high CT-contrast and excellent stability in vivo over a period of 12 weeks. The nanogel is found to be biocompatible and well tolerated. No induction of the inflammatory cytokines INF-γ, IL-6, or TNF-α is observed throughout the study period. The developed nanogel seems to be a safe injectable fiducial marker ideally suited for IGRT that may further enhance the effect of radiation.

Caveolae-mediated endocytosis of intratracheally instilled gold colloid nanoparticles at the air-blood barrier in mice.

Endocytosis is the primary mechanism by which nanoparticles are translocated over the alveolar epithelium. The purpose of this study was to elucidate the association between endocytosis and the translocation of nanoparticles at the air-blood barrier (ABB). Gold colloid particles (diameter, 20 nm) were intratracheally instilled into male ICR mice. Fifteen minutes after instillation, localized accumulation of agglomerated gold particles was observed in the cytoplasm of macrophages, on the surface of alveolar epithelial cells (AECs), and in alveoli. Electron microscopy revealed particles in the vesicles of macrophages, on the surface of AECs, and in caveolae-like vesicles in type 1 AECs. Immunohistochemistry demonstrated positive immunolabeling for caveolin-1 in the ABB of untreated lungs as well as lungs treated with gold particles. Double immunofluorescence and immunoelectron microscopy revealed the presence of caveolin-1 in AECs in the untreated lungs. These results suggest that instilled gold colloid particles are internalized into the alveolar epithelium at the ABB by caveolae-mediated endocytosis, which is regarded as a physiological function of AECs.

Identification of anomalous features of intravitreal injections using micro-computed tomography.

PURPOSE: To identify anomalous features that impact drug delivery in the eye as a result of intravitreal injections using micro-computed tomography imaging. METHODS: Three-dimensional micro-computed tomography images were acquired following an intravitreal injection of 0.03 mL of contrast agent into ex vivo porcine eyes (n = 24). A baseline scan was acquired prior to injection to detect any abnormalities in the eyes. Acquisition continued at various time intervals up to 230 min post-injection. RESULTS: Air bubbles were clearly visible within the vitreous of 21 eyes following injections. There was a total of 36 air bubbles in the 21 eyes and the volume of the air bubbles ranged from 0.01 µL to 1.50 µL. It was found the size of the air bubbles decreased over the scanning period. Furthermore, many of the injected boli in the eye specimens did not have the commonly assumed spherical shape; rather, a variety of other shapes resulted. CONCLUSION: The presence of air bubbles and inconsistent bolus shapes have indicated that intravitreal injections have high variability. It is only through the realization of these anomalous features that the efficacy of intravitreal drug delivery will be improved through a consistent and accurate injection technique.

In situ monitoring of adipogenesis with human-adipose-derived stem cells using surface-enhanced Raman spectroscopy.

Methods capable of nondestructively collecting high-quality, real-time chemical information from living human stem cells are of increasing importance given the escalating relevance of stem cells in therapeutic and regenerative medicines. Raman spectroscopy is one such technique that can nondestructively collect real-time chemical information. Living cells uptake gold nanoparticles and transport these particles through an endosomal pathway. Once inside the endosome, nanoparticles aggregate into clusters that give rise to large spectroscopic enhancements that can be used to elucidate local chemical environments through the use of surface-enhanced Raman spectroscopy. This report uses 40-nm colloidal gold nanoparticles to create volumes of surface-enhanced Raman scattering (SERS) within living human-adipose-derived adult stem cells enabling molecular information to be monitored. We exploit this method to spectroscopically observe chemical changes that occur during the adipogenic differentiation of human-adipose-derived stem cells over a period of 22 days. It is shown that intracellular SERS is able to detect the production of lipids as little as one day after the onset of adipogenesis and that a complex interplay between lipids, proteins, and chemical messengers can be observed shortly thereafter. After 22 days of differentiation, the cells show visible and spectroscopic indications of completed adipogenesis yet still share spectral features common to the progenitor stem cells.

Phase I and pharmacokinetic studies of CYT-6091, a novel PEGylated colloidal gold-rhTNF nanomedicine.

PURPOSE: A novel nanomedicine, CYT-6091, constructed by simultaneously binding recombinant human tumor necrosis factor alpha (rhTNF) and thiolyated polyethylene glycol to the surface of 27-nm colloidal gold particles, was tested in a phase I dose escalation clinical trial in advanced stage cancer patients. EXPERIMENTAL DESIGN: CYT-6091, whose dosing was based on the amount of rhTNF in the nanomedicine, was injected intravenously, and 1 cycle of treatment consisted of 2 treatments administered 14 days apart. RESULTS: Doses from 50 µg/m(2) to 600 µg/m(2) were well tolerated, and no maximum tolerated dose (MTD) was reached, as the highest dose exceeded the target dosage of 1-mg rhTNF per treatment, exceeding the previous MTD for native rhTNF by 3-fold. The first 2 patients on the study, each receiving 50 µg/m(2), did not receive any prophylactic antipyretics or H2 blockade. A predicted, yet controllable fever occurred in these patients, so all subsequently treated patients received prophylactic antipyretics and H2 blockers. However, even at the highest dose rhTNF's dose-limiting toxic effect of hypotension was not seen. Using electron microscopy to visualize nanoparticles of gold in patient biopsies of tumor and healthy tissue showed that patient biopsies taken 24 hours after treatment had nanoparticles of gold in tumor tissue. CONCLUSIONS: These data indicate that rhTNF formulated as CYT-6091 may be administered systemically at doses of rhTNF that were previously shown to be toxic and that CYT-6091 may target to tumors. Future clinical studies will focus on combining CYT-6091 with approved chemotherapies for the systemic treatment of nonresectable cancers.

Fenestrations and preferential flow routes in the prelaminar optic nerve through wet scanning electron microscope and perfusion of tracers.

PURPOSE: We study the vitreous interface of the optic disc to delimit the passages for the flow of fluids through the prelaminar tissue of porcine eyes. METHODS: Wet scanning electron microscope (SEM), conventional SEM and transmission electron microscope (TEM) were used to explore the surface of the optic nerve of the pig. The vitreous cavity was perfused with a fluorescent marker and colloidal gold at controlled pressure. Samples of perfused optic nerve head were cryosectioned and observed with the confocal laser microscope (lectin) or resin embedded and observed under TEM (gold). RESULTS: Fenestrations were present under the SEM in all three regions of the vitreous interface. SEM results were confirmed at the TEM level and under the wet-SEM. Perfusion experiments traced the flow of a fluorescent molecule delineating routes of preferential flow with origin in the fenestrations. Colloidal gold marked the site of entrance in the prelaminar tissue identifying major fenestrations in the basal membrane. CONCLUSIONS: Interchange of fluid between the optic nerve and the vitreous cavity in the pig is facilitated by fenestrations of varied sizes in the basal membrane and preferential flow routes through the prelaminar tissue. Preferential flow routes exist in the extracellular spaces of Elschnig and Kuhn' astrocytes and give a sharply distinct image when compared with flow through zones in which astrocytes envelope axons. Escape routes may be instrumental in preventing oedemas of the optic nerve head, but they could also serve as entrance doors for fluids from the vitreous and aqueous and play a pathogenic role in ageing and glaucoma.

Optical probes for biological applications based on surface-enhanced Raman scattering from indocyanine green on gold nanoparticles.

We report surface-enhanced Raman scattering (SERS) studies on indocyanine green (ICG) on colloidal silver and gold and demonstrate a novel optical probe for applications in living cells. In addition to its own detection by the characteristic ICG SERS signatures, the ICG gold nanoprobe delivers spatially localized chemical information from its biological environment by employing SERS in the local optical fields of the gold nanoparticles. The probe offers the potential to increase the spectral specificity and selectivity of current chemical characterization approaches of living cells and biomaterials based on vibrational information.

The use of gold nanoparticles to enhance radiotherapy in mice.

Mice bearing subcutaneous EMT-6 mammary carcinomas received a single intravenous injection of 1.9 nm diameter gold particles (up to 2.7 g Au/kg body weight), which elevated concentrations of gold to 7 mg Au/g in tumours. Tumour-to-normal-tissue gold concentration ratios remained approximately 8:1 during several minutes of 250 kVp x-ray therapy. One-year survival was 86% versus 20% with x-rays alone and 0% with gold alone. The increase in tumours safely ablated was dependent on the amount of gold injected. The gold nanoparticles were apparently non-toxic to mice and were largely cleared from the body through the kidneys. This novel use of small gold nanoparticles permitted achievement of the high metal content in tumours necessary for significant high-Z radioenhancement.

Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery.

Colloidal gold, a sol comprised of nanoparticles of Au(0), has been used as a therapeutic for the treatment of cancer as well as an indicator for immunodiagnostics. However, the use of these gold nanoparticles for in vivo drug delivery has never been described. This communication outlines the development of a colloidal gold (cAu) nanoparticle vector that targets the delivery of tumor necrosis factor (TNF) to a solid tumor growing in mice. The optimal vector, designated PT-cAu-TNF, consists of molecules of thiol-derivatized PEG (PT) and recombinant human TNF that are directly bound onto the surface of the gold nanoparticles. Following intravenous administration, PT-cAu-TNF rapidly accumulates in MC-38 colon carcinoma tumors and shows little to no accumulation in the livers, spleens (i.e., the RES) or other healthy organs of the animals. The tumor accumulation was evidenced by a marked change in the color of the tumor as it acquired the bright red/purple color of the colloidal gold sol and was coincident with the active and tumor-specific sequestration of TNF. Finally, PT-cAu-TNF was less toxic and more effective in reducing tumor burden than native TNF since maximal antitumor responses were achieved at lower doses of drug.

Enhancement of the effectiveness of electroporation-augmented cutaneous DNA vaccination by a particulate adjuvant.

DNA vaccines are attracting increased attention due to multiple advantages over conventional vaccines. Attempts to improve these vaccines focus on enhancing DNA delivery and employing novel immunoadjuvants. Electroporation (EP) has emerged as an effective method for delivering DNA vaccines, significantly enhancing humoral and cellular responses. To further improve EP-augmented DNA vaccination, we used micron-size gold particles as a particulate adjuvant. DNA is not bound, or adsorbed, to the particles. Gold particles were coinjected intradermally with plasmid DNA encoding the hepatitis B virus surface antigen (HBsAg) into mice, both in the absence and presence of noninvasive EP. The particles enhanced the percentage of responding animals, and shortened the time for reaching maximal antibody titers by 2 weeks. Subtyping of the produced antibodies revealed a predominantly Th1-like response which did not change significantly with the absence or presence of particles. The particles likely function as an attractant for antigen-presenting cells (APCs), and probably do not affect EP or antigen expression to a significant extent. We conclude that micron-size gold particles injected intradermally together with DNA followed by EP give rise to an accelerated, potent immune response with a strong cellular component. This method may become important for the development of fast-acting therapeutic and prophylactic vaccines.

Transport of nitrated albumin across continuous vascular endothelium.

Because modification of plasma albumin on tyrosine residues generates nitrated albumin (NOA) that may function as a mechanism of nitrogen monoxide clearance from microcirculation, we investigated biochemicaly and morphologically the cell surface binding and the transendothelial transport of NOA. An electron microscopic study was carried out with mouse lungs and hearts perfused in situ with NOA and NOA-Au complexes. The results indicate that NOA-Au can bind to the endothelial cell surface, and its binding can be blocked by albumin plus nitrotyrosine (NO-tyrosine) or abolished by excess NOA. We detected NOA-Au into perivascular spaces as early as 30 sec after the beginning of its perfusion. NOA, unlike native albumin, leaves the vascular lumina via both endothelial caveolae and open junctions. By cross-linking and ligand blotting analysis, we showed that NOA interacted with the same albumin binding proteins of 16-18, 30-32, 60, and 74 kDa as native albumin. ELISA performed on tissue homogenates obtained from the same specimens showed that NOA transport was 2- to 4-fold greater than native albumin. The augmented transendothelial transport of NOA reflects its transcytosis as well as its exit from the microcirculation via open junctions. The increased transport of NOA may serve as an important mechanism that protects a vascular bed against the damaging effects of nitrogen monoxide and peroxynitrite.

Gastrointestinal persorption and tissue distribution of differently sized colloidal gold nanoparticles.

The gastrointestinal uptake of micro- and nanoparticles has been the subject of recent efforts to develop effective carriers that enhance the oral uptake of drugs and vaccines. Here, we used correlative instrumental neutron activation analysis and electron microscopy to quantitatively and qualitatively study the gastrointestinal uptake and subsequent tissue/organ distribution of 4, 10, 28, and 58 nm diameter metallic colloidal gold particles following oral administration to mice. In our quantitative studies we found that colloidal gold uptake is dependent on particle size: smaller particles cross the gastrointestinal tract more readily. Electron microscopic studies showed that particle uptake occurred in the small intestine by persorption through single, degrading enterocytes in the process of being extruded from a villus. To our knowledge this is the first report, at the ultrastructural level, of gastrointestinal uptake of particulates by persorption through holes created by extruding enterocytes.

Medullary neurones regulate hypothalamic corticotropin-releasing factor cell responses to an emotional stressor.

Hypothalamic-pituitary-adrenal axis activation is a hallmark of the stress response. In the case of physical stressors, there is considerable evidence that medullary catecholamine neurones are critical to the activation of the paraventricular nucleus corticotropin-releasing factor cells that constitute the apex of the hypothalamic-pituitary-adrenal axis. In contrast, it has been thought that hypothalamic-pituitary-adrenal axis responses to emotional stressors do not involve brainstem neurones. To investigate this issue we have mapped patterns of restraint-induced neuronal c-fos expression in intact animals and in animals prepared with either paraventricular nucleus-directed injections of a retrograde tracer, lesions of paraventricular nucleus catecholamine terminals, or lesions of the medulla corresponding to the A1 or A2 noradrenergic cell groups. Restraint-induced patterns of neuronal activation within the medulla of intact animals were very similar to those previously reported in response to physical stressors, including the fact that most stressor-responsive, paraventricular nucleus-projecting cells were certainly catecholaminergic and probably noradrenergic. Despite this, the destruction of paraventricular nucleus catecholamine terminals with 6-hydroxydopamine did not alter corticotropin-releasing factor cell responses to restraint. However, animals with ibotenic acid lesions encompassing either the A1 or A2 noradrenergic cell groups displayed significantly suppressed corticotropin-releasing factor cell responses to restraint. Notably, these medullary lesions also suppressed neuronal responses in the medial amygdala, an area that is now considered critical to hypothalamic-pituitary-adrenal axis responses to emotional stressors and that is also known to display a significant increase in noradrenaline turnover during restraint. We conclude that medullary neurones influence corticotropin-releasing factor cell responses to emotional stressors via a multisynaptic pathway that may involve a noradrenergic input to the medial amygdala. These results overturn the idea that hypothalamic-pituitary-adrenal axis response to emotional stressors can occur independently of the brainstem.

Host factors influencing the preferential localization of sterically stabilized liposomes in Klebsiella pneumoniae-infected rat lung tissue.

PURPOSE: To gain insight into the host factors influencing liposome localization at sites of bacterial infection. METHODS: In a unilateral Klebsiella pneumoniae pneumonia rat model, capillary permeability and number of circulating leukocytes was quantified and related to the degree of liposome target localization. RESULTS: Liposome localization was highest in the hemorrhagic zone of infection, a zone characterized by markedly increased capillary permeability and high bacterial numbers. Both liposome localization and capillary permeability correlated positively with severity of infection. Lung instillation of other inflammatory stimuli, such as lipopolysaccharide or 0.1 M HCl inducing increased capillary permeability, also promoted liposome localization. As liposomal target localization in leukopenic rats was similar to that in immunocompetent rats, contribution of circulating leukocytes seems limited. Intrapulmonary distribution of liposomes shows that leukocytes at the target site are involved in liposome uptake after extravasation. CONCLUSIONS: Increased capillary permeability plays a crucial role in liposome localization at the infected site, whereas contribution of leukocytes is limited. These results suggest inflammatory conditions that could benefit from liposomal drug delivery. The involvement of leukocytes in liposome uptake at the target site could be important information in the selection of appropriate drugs.

Dendrimer-assisted patch-clamp sizing of nuclear pores.

Macromolecular translocation (MMT) across the nuclear envelope (NE) occurs exclusively through the nuclear pore complex (NPC). Therefore, the diameter of the NPC aqueous/electrolytic channel (NPCC) is important for cellular structure and function. The NPCC diameter was previously determined to be approximately equal to 10 nm with electron microscopy (EM) using the translocation of colloidal gold particles. Here we present patch-clamp and fluorescence microscopy data from adult cardiomyocyte nuclei that demonstrate the use of patch-clamp for assessing NPCC diameter. Fluorescence microscopy with B-phycoerythrin (BPE, 240 kDa) conjugated to a nuclear localization signal (NLS) demonstrated that these nuclei were competent for NPC-mediated MMT (NPC-MMT). Furthermore, when exposed to an appropriate cell lysate, the nuclei expressed enhanced green fluorescence protein (EGFP) after 5-10 h of incubation with the plasmid for this protein (pEGFP, 3.1 MDa). Nucleus-attached patch-clamp showed that colloidal gold particles were not useful probes; they modified NPCC gating. As a result of this finding, we searched for an inert class of particles that could be used without irreversibly affecting NPCC gating and found that fluorescently labeled Starburst dendrimers, a distinct class of polymers, were useful. Our patch-clamp and fluorescence microscopy data with calibrated dendrimers indicate that the cardiomyocyte NPCC diameter varies between 8 and 9 nm. These studies open a new direction in the investigation of live, continuous NPC dynamics under physiological conditions.