A semiconductor 96-microplate platform for electrical-imaging based high-throughput phenotypic screening.

High-content imaging for compound and genetic profiling is popular for drug discovery but limited to endpoint images of fixed cells. Conversely, electronic-based devices offer label-free, live cell functional information but suffer from limited spatial resolution or throughput. Here, we introduce a semiconductor 96-microplate platform for high-resolution, real-time impedance imaging. Each well features 4096 electrodes at 25 µm spatial resolution and a miniaturized data interface allows 8× parallel plate operation (768 total wells) for increased throughput. Electric field impedance measurements capture >20 parameter images including cell barrier, attachment, flatness, and motility every 15 min during experiments. We apply this technology to characterize 16 cell types, from primary epithelial to suspension cells, and quantify heterogeneity in mixed co-cultures. Screening 904 compounds across 13 semiconductor microplates reveals 25 distinct responses, demonstrating the platform's potential for mechanism of action profiling. The scalability and translatability of this semiconductor platform expands high-throughput mechanism of action profiling and phenotypic drug discovery applications.

A semiconductor 96-microplate platform for electrical-imaging based high-throughput phenotypic screening.

Profiling compounds and genetic perturbations via high-content imaging has become increasingly popular for drug discovery, but the technique is limited to endpoint images of fixed cells. In contrast, electronic-based devices offer label-free, functional information of live cells, yet current approaches suffer from low-spatial resolution or single-well throughput. Here, we report a semiconductor 96-microplate platform designed for high-resolution real-time impedance "imaging" at scale. Each well features 4,096 electrodes at 25 µm spatial resolution while a miniaturized data interface allows 8× parallel plate operation (768 total wells) within each incubator for enhanced throughputs. New electric field-based, multi-frequency measurement techniques capture >20 parameter images including tissue barrier, cell-surface attachment, cell flatness, and motility every 15 min throughout experiments. Using these real-time readouts, we characterized 16 cell types, ranging from primary epithelial to suspension, and quantified heterogeneity in mixed epithelial and mesenchymal co-cultures. A proof-of-concept screen of 904 diverse compounds using 13 semiconductor microplates demonstrates the platform's capability for mechanism of action (MOA) profiling with 25 distinct responses identified. The scalability of the semiconductor platform combined with the translatability of the high dimensional live-cell functional parameters expands high-throughput MOA profiling and phenotypic drug discovery applications.

Protective immunity enhanced Salmonella vaccine vectors delivering Helicobacter pylori antigens reduce H. pylori stomach colonization in mice.

Helicobacter pylori is a major cause of gastric mucosal inflammation, peptic ulcers, and gastric cancer. Emerging antimicrobial-resistant H. pylori has hampered the effective eradication of frequent chronic infections. Moreover, a safe vaccine is highly demanded due to the absence of effective vaccines against H. pylori. In this study, we employed a new innovative Protective Immunity Enhanced Salmonella Vaccine (PIESV) vector strain to deliver and express multiple H. pylori antigen genes. Immunization of mice with our vaccine delivering the HpaA, Hp-NAP, UreA and UreB antigens, provided sterile protection against H. pylori SS1 infection in 7 out of 10 tested mice. In comparison to the control groups that had received PBS or a PIESV carrying an empty vector, immunized mice exhibited specific and significant cellular recall responses and antigen-specific serum IgG1, IgG2c, total IgG and gastric IgA antibody titers. In conclusion, an improved S. Typhimurium-based live vaccine delivering four antigens shows promise as a safe and effective vaccine against H. pylori infection.

CMOS electrochemical pH localizer-imager.

pH controls a large repertoire of chemical and biochemical processes in water. Densely arrayed pH microenvironments would parallelize these processes, enabling their high-throughput studies and applications. However, pH localization, let alone its arrayed realization, remains challenging because of fast diffusion of protons in water. Here, we demonstrate arrayed localizations of picoliter-scale aqueous acids, using a 256-electrochemical cell array defined on and operated by a complementary metal oxide semiconductor (CMOS)-integrated circuit. Each cell, comprising a concentric pair of cathode and anode with their current injections controlled with a sub-nanoampere resolution by the CMOS electronics, creates a local pH environment, or a pH "voxel," via confined electrochemistry. The system also monitors the spatiotemporal pH profile across the array in real time for precision pH control. We highlight the utility of this CMOS pH localizer-imager for high-throughput tasks by parallelizing pH-gated molecular state encoding and pH-regulated enzymatic DNA elongation at any selected set of cells.

Multi-parametric functional imaging of cell cultures and tissues with a CMOS microelectrode array.

Electrode-based impedance and electrochemical measurements can provide cell-biology information that is difficult to obtain using optical-microscopy techniques. Such electrical methods are non-invasive, label-free, and continuous, eliminating the need for fluorescence reporters and overcoming optical imaging's throughput/temporal resolution limitations. Nonetheless, electrode-based techniques have not been heavily employed because devices typically contain few electrodes per well, resulting in noisy aggregate readouts. Complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs) have sometimes been used for electrophysiological measurements with thousands of electrodes per well at sub-cellular pitches, but only basic impedance mappings of cell attachment have been performed outside of electrophysiology. Here, we report on new field-based impedance mapping and electrochemical mapping/patterning techniques to expand CMOS-MEA cell-biology applications. The methods enable accurate measurement of cell attachment, growth/wound healing, cell-cell adhesion, metabolic state, and redox properties with single-cell spatial resolution (20 µm electrode pitch). These measurements allow the quantification of adhesion and metabolic differences of cells expressing oncogenes versus wild-type controls. The multi-parametric, cell-population statistics captured by the chip-scale integrated device opens up new avenues for fully electronic high-throughput live-cell assays for phenotypic screening and drug discovery applications.

Therapeutic effects of equine amniotic membrane suspension on corneal re-epithelialization and haze in a modified lagomorph ex vivo wound healing model.

OBJECTIVE: To investigate the therapeutic effects of topical equine amniotic membrane (eAM) suspension following corneal wounding in a controlled experimental setting. PROCEDURES: Equine amniotic membrane was collected, gamma irradiated, homogenized for topical suspension preparation, and cryopreserved. Corneoscleral rims harvested from fresh rabbit globes were wounded via keratectomy and were maintained in an air-liquid interface ex vivo corneal culture model. Treatment groups included topical gamma irradiated eAM suspension (n = 20) and a control group (n = 20). Re-epithelialization of the wound was assessed with daily photographic evaluation of area of fluorescein uptake (mm2 ). Corneal wound haze after a 21-day period was assessed by photographic analysis of haze area (mm2 ) and pixel intensity (0-255). Histologic processing of corneal tissue was performed, and protein identification of eAM suspension using Liquid chromatography-mass spectrometry (LC-MS). RESULTS: The average day of complete corneal re-epithelialization in controls (5.5 ± 1.1) and topically treated (5.5 ± 0.6) corneas, and rates of reduction in area of fluorescein uptake over time did not significantly differ (p = .44). The corneal wound haze was significantly reduced in mean area by approximately 52% and intensity by 57% in corneas treated with topical eAM suspension (p < .05), compared to controls 21 days following wounding. Protein analysis identified numerous proteins, specifically decorin, dermatopontin, and lumican, which have previously been documented in eAM. CONCLUSIONS: Area and intensity of corneal wound haze were significantly reduced in corneas treated with gamma irradiated eAM suspension, which may be due to previously identified therapeutic proteins which promote corneal clarity.

Stimulation of regulatory T cells with Lactococcus lactis expressing enterotoxigenic E. coli colonization factor antigen 1 retains salivary flow in a genetic model of Sjögren's syndrome.

BACKGROUND: Sjögren's syndrome (SjS), one of the most common autoimmune diseases, impacts millions of people annually. SjS results from autoimmune attack on exocrine (salivary and lacrimal) glands, and women are nine times more likely to be affected than men. To date, no vaccine or therapeutic exists to treat SjS, and patients must rely on lifelong therapies to alleviate symptoms. METHODS: Oral treatment with the adhesin from enterotoxigenic Escherichia coli colonization factor antigen I (CFA/I) fimbriae protects against several autoimmune diseases in an antigen (Ag)-independent manner. Lactococcus lactis, which was recently adapted to express CFA/I fimbriae (LL-CFA/I), effectively suppresses inflammation by the induction of infectious tolerance via Ag-specific regulatory T cells (Tregs), that produce IL-10 and TGF-ß. To test the hypothesis that CFA/I fimbriae can offset the development of inflammatory T cells via Treg induction, oral treatments with LL-CFA/I were performed on the spontaneous, genetically defined model for SjS, C57BL/6.NOD-Aec1Aec2 mice to maintain salivary flow. RESULTS: Six-week (wk)-old C57BL/6.NOD-Aec1Aec2 mice were orally dosed with LL-CFA/I and treated every 3 wks; control groups were given L. lactis vector or PBS. LL-CFA/I-treated mice retained salivary flow up to 28 wks of age and showed significantly reduced incidence of inflammatory infiltration into the submandibular and lacrimal glands relative to PBS-treated mice. A significant increase in Foxp3+ and IL-10- and TGF-ß-producing Tregs was observed. Moreover, LL-CFA/I significantly reduced the expression of proinflammatory cytokines, IL-6, IL-17, GM-CSF, and IFN-γ. Adoptive transfer of CD4+ T cells from LL-CFA/I-treated, not LL vector-treated mice, restored salivary flow in diseased SjS mice. CONCLUSION: These data demonstrate that oral LL-CFA/I reduce or halts SjS progression, and these studies will provide the basis for future testing in SjS patients.

Retrospective analysis of ocular disease in a population of captive pteropodid bats, 2003-2020.

OBJECTIVE: To perform retrospective analysis of captive pteropodid bats presented to the University of Florida for ocular or adnexal disease from 2003-2020. ANIMALS STUDIED: Twenty-four individuals from seven species were included. PROCEDURES: Records were analyzed for disease process, methods of treatment, and surgical techniques and complications. RESULTS: The most frequently reported abnormality was corneal disease (79%), followed by cataracts (54%), and uveitis (42%). Corneal disease was primarily attributed to either trauma or exposure keratitis secondary to buphthalmia. The majority of uveitis appeared to be lens-induced. Five cases (21%) of glaucoma were reported, all of which accompanied lens luxation. Of the seven enucleations performed, six had post-operative complications (85.7%), including swelling at the surgical site, seroma formation, and bacterial infection. There was no significant relationship between age and trauma, age and cataract formation, sex and trauma, or species and cataract formation. CONCLUSIONS: The most common underlying cause of ocular pathology in these cases was trauma. While the bats tolerated topical and systemic treatment well, individual temperament must be taken into account when developing treatment plans, and prevention of injury is the most effective management strategy.

Retrobulbar embryonal tumor with multilayered rosettes in a golden retriever dog.

Although rare, embryonal tumors (previously called primitive neuroectodermal tumors) should be considered in the differential diagnosis of retrobulbar tumors in dogs regardless of the age of the patient, and ancillary tests are required for definitive diagnosis.

Pulmonary Sarcomatoid Carcinoma Associated with Arterial Thromboembolism in a Cat.

A 14-year-old, neutered male domestic shorthair cat presented for acute monoparesis with physical exam findings and biochemical data supportive of a distal arterial thromboembolism. Thoracic radiographs revealed an alveolar pattern in the right middle lung lobe and multifocal nodules in other lung lobes. A pulmonary mass was found on necropsy, which was composed of both carcinomatous and sarcomatous components, confirmed with cytokeratin and vimentin immunohistochemistry. Using the World Health Organization classification scheme for mixed pulmonary tumors, this tumor would be characterized as a pleomorphic squamous cell carcinoma under the umbrella term of pulmonary sarcomatoid carcinoma. The World Health Organization classification of mixed pulmonary tumors and its application to previously reported mixed pulmonary tumors in companion animals is discussed. This is the first reported case of this tumor type in a cat, as well as the first report of this tumor type associated with an arterial thromboembolism in any veterinary species.

Laparoscopic gonadectomy in a dog with 78,XX/78,XY chimerism and underdeveloped reproductive organs.

CASE DESCRIPTION: A 1-year-old externally sexually intact female Great Dane was referred for further evaluation of abnormal and underdeveloped internal reproductive organs. CLINICAL FINDINGS: Physical examination findings included a cranioventrally displaced vulva and a grade 2/6 left apical systolic heart murmur. No uterus or ovaries were identified during abdominal ultrasonography. Computed tomography with retrograde vaginourethrography revealed an underdeveloped uterus and possible left intra-abdominal gonad. Karyotyping revealed mixed sex chromosomes (70% XY and 30% XX). Analysis of a serum sample yielded positive results for anti-Müllerian hormone; other findings included mid range estradiol concentration (48.2 pg/mL [within reference intervals for sexually intact and neutered males and females]), low progesterone concentration (< 0.2 ng/mL [within reference intervals for anestrous females]), and low testosterone concentration (< 20 ng/dL [similar to the expected concentration in neutered males]). Overall, the results of the sex hormone analyses were consistent with findings for either a sexually intact female or a neutered male dog. The dog's cardiac structure and function were echocardiographically normal. TREATMENT AND OUTCOME: The dog was anesthetized and underwent laparoscopic gonadectomy. The gonads, although abnormal and underdeveloped, were readily identified intraoperatively and successfully removed. On the basis of histologic findings, the removed gonads were confirmed to be rudimentary testicles. The dog recovered from anesthesia and surgery without complications. CLINICAL RELEVANCE: Laparoscopic surgery was effective for visualization of abnormal and hypoplastic reproductive organs when abdominal ultrasonography and CT were of limited diagnostic usefulness, and laparoscopic surgery allowed straightforward gonadectomy in a 78,XX/78,XY chimeric dog.

Extracellular recording of direct synaptic signals with a CMOS-nanoelectrode array.

The synaptic connections between neurons are traditionally determined by correlating the action potentials (APs) of a pre-synaptic neuron and small-amplitude subthreshold potentials of a post-synaptic neuron using invasive intracellular techniques, such as patch clamping. Extracellular recording by a microelectrode array can non-invasively monitor network activities of a large number of neurons, but its reduced sensitivity usually prevents direct measurements of synaptic signals. Here, we demonstrate that a newly developed complementary metal-oxide-semiconductor (CMOS) nanoelectrode array (CNEA) is capable of extracellularly determining direct synaptic connections in dense, multi-layer cultures of dissociated rat neurons. We spatiotemporally correlate action potential signals of hundreds of active neurons, detect small (∼1 pA after averaging) extracellular synaptic signals at the region where pre-synaptic axons and post-synaptic dendrites/somas overlap, and use those signals to map synaptic connections. We use controlled stimulation to assess stimulation-dependent synaptic strengths and to titrate a synaptic blocker (CNQX: IC50 ∼ 1 µM). The new capabilities demonstrated here significantly enhance the utilities of CNEAs in connectome mapping and drug screening applications.

Oral therapy with colonization factor antigen I prevents development of type 1 diabetes in Non-obese Diabetic mice.

Antigen (Ag)-specific tolerization prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice but proved less effective in humans. Several auto-Ags are fundamental to disease development, suggesting T1D etiology is heterogeneous and may limit the effectiveness of Ag-specific therapies to distinct disease endotypes. Colonization factor antigen I (CFA/I) fimbriae from Escherichia coli can inhibit autoimmune diseases in murine models by inducing bystander tolerance. To test if Ag-independent stimulation of regulatory T cells (Tregs) can prevent T1D onset, groups of NOD mice were orally treated with Lactococcus lactis (LL) expressing CFA/I. LL-CFA/I treatment beginning at 6 weeks of age reduced disease incidence by 50% (p < 0.05) and increased splenic Tregs producing both IL-10 and IFN-γ 8-fold (p < 0.005) compared to LL-vehicle treated controls. To further describe the role of these Tregs in preventing T1D, protective phenotypes were examined at different time-points. LL-CFA/I treatment suppressed splenic TNF-α+CD8+ T cells 6-fold at 11 weeks (p < 0.005) and promoted a distinct microbiome. At 17 weeks, IFN-γ+CD4+ T cells were suppressed 10-fold (p < 0.005), and at 30 weeks, pancreatic Tbet+CD4+ T cells were suppressed (p < 0.05). These results show oral delivery of modified commensal organisms, such as LL-CFA/I, may be harnessed to restrict Th1 cell-mediated immunity and protect against T1D.

The Design of a CMOS Nanoelectrode Array with 4096 Current-Clamp/Voltage-Clamp Amplifiers for Intracellular Recording/Stimulation of Mammalian Neurons.

CMOS microelectrode arrays (MEAs) can record electrophysiological activities of a large number of neurons in parallel but only extracellularly with low signal-to-noise ratio. Patch clamp electrodes can perform intracellular recording with high signal-to-noise ratio but only from a few neurons in parallel. Recently we have developed and reported a neuroelectronic interface that combines the parallelism of the CMOS MEA and the intracellular sensitivity of the patch clamp. Here, we report the design and characterization of the CMOS integrated circuit (IC), a critical component of the neuroelectronic interface. Fabricated in 0.18-µm technology, the IC features an array of 4,096 platinum black (PtB) nanoelectrodes spaced at a 20 µm pitch on its surface and contains 4,096 active pixel circuits. Each active pixel circuit, consisting of a new switched-capacitor current injector--capable of injecting from ±15 pA to ±0.7 µA with a 5 pA resolution--and an operational amplifier, is highly configurable. When configured into current-clamp mode, the pixel intracellularly records membrane potentials including subthreshold activities with ∼23 µVrms input referred noise while injecting a current for simultaneous stimulation. When configured into voltage-clamp mode, the pixel becomes a switched-capacitor transimpedance amplifier with ∼1 pArms input referred noise, and intracellularly records ion channel currents while applying a voltage for simultaneous stimulation. Such voltage/current-clamp intracellular recording/stimulation is a feat only previously possible with the patch clamp method. At the same time, as an array, the IC overcomes the lack of parallelism of the patch clamp method, measuring thousands of mammalian neurons in parallel, with full-frame intracellular recording/stimulation at 9.4 kHz.

A nanoelectrode array for obtaining intracellular recordings from thousands of connected neurons.

Current electrophysiological or optical techniques cannot reliably perform simultaneous intracellular recordings from more than a few tens of neurons. Here we report a nanoelectrode array that can simultaneously obtain intracellular recordings from thousands of connected mammalian neurons in vitro. The array consists of 4,096 platinum-black electrodes with nanoscale roughness fabricated on top of a silicon chip that monolithically integrates 4,096 microscale amplifiers, configurable into pseudocurrent-clamp mode (for concurrent current injection and voltage recording) or into pseudovoltage-clamp mode (for concurrent voltage application and current recording). We used the array in pseudovoltage-clamp mode to measure the effects of drugs on ion-channel currents. In pseudocurrent-clamp mode, the array intracellularly recorded action potentials and postsynaptic potentials from thousands of neurons. In addition, we mapped over 300 excitatory and inhibitory synaptic connections from more than 1,700 neurons that were intracellularly recorded for 19 min. This high-throughput intracellular-recording technology could benefit functional connectome mapping, electrophysiological screening and other functional interrogations of neuronal networks.

Regulating colonic dendritic cells by commensal glycosylated large surface layer protein A to sustain gut homeostasis against pathogenic inflammation.

Microbial interaction with the host through sensing receptors, including SIGNR1, sustains intestinal homeostasis against pathogenic inflammation. The newly discovered commensal Propionibacterium strain, P. UF1, regulates the intestinal immunity against pathogen challenge. However, the molecular events driving intestinal phagocytic cell response, including colonic dendritic cells (DCs), by this bacterium are still elusive. Here, we demonstrate that the glycosylation of bacterial large surface layer protein A (LspA) by protein O-mannosyltransferase 1 (Pmt1) regulates the interaction with SIGNR1, resulting in the control of DC transcriptomic and metabolomic machineries. Programmed DCs promote protective T cell response to intestinal Listeria infection and resist chemically induced colitis in mice. Thus, our findings may highlight a novel molecular mechanism by which commensal surface glycosylation interacting with SIGNR1 directs the intestinal homeostasis to potentially protect the host against proinflammatory signals inducing colonic tissue damage.

Primary leptomeningeal gliomatosis in a domestic shorthaired cat.

A 15-y-old neutered male domestic shorthaired cat was presented with a 16-d history of hindlimb paralysis in conjunction with 1-wk duration of inappetence and lethargy. Given intractable clinical signs, development of seizures, and poor prognosis, euthanasia was elected. Gross examination revealed mild, chronic, multifocal intervertebral disk disease; however, no gross abnormalities were noted in the spinal cord. Histologic examination of the cervical, thoracic, and lumbar spinal cord and the myelencephalon revealed diffuse and variable expansion of the meninges by sheets of neoplastic round-to-polygonal cells. The cells formed sheets and clusters, supported by a variably eosinophilic, fibrillar-to-basophilic, homogeneous matrix, and contained a small amount of eosinophilic cytoplasm. The nuclei were round with finely stippled to hyperchromatic chromatin and 1-2 small nucleoli. Mild white matter degeneration was present in the dorsal and ventral funiculi multifocally throughout the spinal cord, but was most severe in the ventral lumbar sections. Immunohistochemistry revealed strong intranuclear immunoreactivity for Olig2, and intracytoplasmic immunoreactivity for glial fibrillary acidic protein, MAP2, and vimentin in the neoplastic glial cells. To our knowledge, primary leptomeningeal gliomatosis has not been reported previously in a cat.

Anatomic characterization of pulmonary accessory fissures in canine cadavers.

Accessory fissures in the lungs are well described in humans, considered a normal finding, being identified in 60% of autopsied lungs, and more prevalent in the right lung (Gesase, ; Nene, Gajendra, & Sarma, 2011). In dogs, interlobar fissures are well recognized, but there is the lack of anatomic characterization for accessory fissures in the accessible literature. The purpose of this descriptive study was to identify the prevalence and to describe the anatomic localization of accessory fissures. The lungs from 87 dog cadavers used to teach veterinary gross anatomy were collected. Accessory fissures were characterized for each lung lobe, specific lung lobe location, orientation, length and the number of accessory fissures per lobe. Accessory fissures were recognized in 48/87 (55%) of canine lungs, all located in the periphery of the individual lobes. We found a significant association between the presence of accessory fissures and the costal surface of the lung (p < 0.0001), the right lung (p < 0.004), the right cranial lung lobe (p < 0.002) and the left cranial lung lobe (p < 0.04). Histologic results showed normal alveolar and respiratory bronchioles to the level of the accessory fissures. Our results show that accessory fissures are a common finding and should be considered a normal variant. They are more prevalent in the right lung, in the costal surface, and in the right and left cranial lung lobes. An anatomic nomenclature for accessory fissures based on the current Nomina Anatomica Veterinaria and the human literature is proposed. Further studies include a comparison among anatomical accessory fissures, and radiographic and computed tomographic images.

Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology.

Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane, clarifying how the nanoelectrode attains intracellular access. This understanding will be translated into a circuit model for the nanobio interface, which we will then use to lay out the strategies for improving the interface. The intracellular interface of the nanoelectrode is currently inferior to that of the patch clamp electrode; reaching this benchmark will be an exciting challenge that involves optimization of electrode geometries, materials, chemical modifications, electroporation protocols, and recording/stimulation electronics, as we describe in the Account. Another important theme of this Account, beyond the optimization of the individual nanoelectrode-cell interface, is the scalability of the nanoscale electrodes. We will discuss this theme using a recent development from our groups as an example, where an array of ca. 1000 nanoelectrode pixels fabricated on a CMOS integrated circuit chip performs parallel intracellular recording from a few hundreds of cardiomyocytes, which marks a new milestone in electrophysiology.