Hepatocytes Research Articles

Single-Cell Microfluidics: A Primer for Microbiologists.

Recent advances in microfluidic technology have made it possible to image live bacterial cells with a high degree of precision and control. In particular, single-cell microfluidic designs have created new opportunities to study phenotypic variation in bacterial populations. However, the development and use of microfluidic devices require specialized resources, and these can be practical barriers to entry for microbiologists. With this review, our intentions are to help demystify the design, construction, and application of microfluidics. Our approach is to present design elements as building blocks from which a multitude of microfluidics applications can be imagined by the microbiologist.

Ultrasensitive Electrochemical Detection of Salmonella typhimurium in Food Matrices Using Surface-Modified Bacterial Cellulose with Immobilized Phage Particles.

The rapid and sensitive detection of Salmonella typhimurium in food matrices is crucial for ensuring food safety. This study presents the development of an ultrasensitive electrochemical biosensor using surface-modified bacterial cellulose (BC) integrated with polypyrrole (Ppy) and reduced graphene oxide (RGO), further functionalized with immobilized S. typhimurium-specific phage particles. The BC substrate, with its ultra-fibrous and porous structure, was modified through in situ oxidative polymerization of Ppy and RGO, resulting in a highly conductive and flexible biointerface. The immobilization of phages onto this composite was facilitated by electrostatic interactions between the polycationic Ppy and the negatively charged phage capsid heads, optimizing phage orientation and enhancing bacterial capture efficiency. Morphological and chemical characterization confirmed the successful fabrication and phage immobilization. The biosensor demonstrated a detection limit of 1 CFU/mL for S. typhimurium in phosphate-buffered saline (PBS), with a linear detection range spanning 100 to 107 CFU/mL. In real samples, the sensor achieved detection limits of 5 CFU/mL in milk and 3 CFU/mL in chicken, with a linear detection range spanning 100 to 106 CFU/mL, maintaining high accuracy and reproducibility. The biosensor also effectively discriminated between live and dead bacterial cells, demonstrating its potential in real-world food safety applications. The biosensor performed excellently over a wide pH range (4-10) and remained stable for up to six weeks. Overall, the developed BC/Ppy/RGO-phage biosensor offers a promising tool for the rapid, sensitive, and selective detection of S. typhimurium, with robust performance across different food matrices.

Physiological liver microtissue 384-well microplate system for preclinical hepatotoxicity assessment of therapeutic small molecule drugs.

Hepatotoxicity can lead to the discontinuation of approved or investigational drugs. The evaluation of the potential hepatoxicity of drugs in development is challenging because current models assessing this adverse effect are not always predictive of the outcome in human beings. Cell lines are routinely used for early hepatotoxicity screening, but to improve the detection of potential hepatotoxicity, in vitro models that better reflect liver morphology and function are needed. One such promising model is human liver microtissues. These are spheroids made of primary human parenchymal and nonparenchymal liver cells, which are amenable to high throughput screening. To test the predictivity of this model, the cytotoxicity of 152 FDA (US Food & Drug Administration)-approved small molecule drugs was measured as per changes in ATP content in human liver microtissues incubated in 384-well microplates. The results were analyzed with respect to drug label information, drug-induced liver injury (DILI) concern class, and drug class. The threshold IC50ATP-to-Cmax ratio of 176 was used to discriminate between safe and hepatotoxic drugs. "vMost-DILI-concern" drugs were detected with a sensitivity of 72% and a specificity of 89%, and "vMost-DILI-concern" drugs affecting the nervous system were detected with a sensitivity of 92% and a specificity of 91%. The robustness and relevance of this evaluation were assessed using a 5-fold cross-validation. The good predictivity, together with the in vivo-like morphology of the liver microtissues and scalability to a 384-well microplate, makes this method a promising and practical in vitro alternative to 2D cell line cultures for the early hepatotoxicity screening of drug candidates.

A sphingolipid rheostat controls apoptosis versus apical cell extrusion as alternative tumour-suppressive mechanisms

Evasion of cell death is a hallmark of cancer, and consequently the induction of cell death is a common strategy in cancer treatment. However, the molecular mechanisms regulating different types of cell death are poorly understood. We have formerly shown that in the epidermis of hypomorphic zebrafish hai1a mutant embryos, pre-neoplastic transformations of keratinocytes caused by unrestrained activity of the type II transmembrane serine protease Matriptase-1 heal spontaneously. This healing is driven by Matriptase-dependent increased sphingosine kinase (SphK) activity and sphingosine-1-phosphate (S1P)-mediated keratinocyte loss via apical cell extrusion. In contrast, amorphic hai1afr26 mutants with even higher Matriptase-1 and SphK activity die within a few days. Here we show that this lethality is not due to epidermal carcinogenesis, but to aberrant tp53-independent apoptosis of keratinocytes caused by increased levels of pro-apoptotic C16 ceramides, sphingolipid counterparts to S1P within the sphingolipid rheostat, which severely compromises the epidermal barrier. Mathematical modelling of sphingolipid rheostat homeostasis, combined with in vivo manipulations of components of the rheostat or the ceramide de novo synthesis pathway, indicate that this unexpected overproduction of ceramides is caused by a negative feedback loop sensing ceramide levels and controlling ceramide replenishment via de novo synthesis. Therefore, despite their initial decrease due to increased conversion to S1P, ceramides eventually reach cell death-inducing levels, making transformed pre-neoplastic keratinocytes die even before they are extruded, thereby abrogating the normally barrier-preserving mode of apical live cell extrusion. Our results offer an in vivo perspective of the dynamics of sphingolipid homeostasis and its relevance for epithelial cell survival versus cell death, linking apical cell extrusion and apoptosis. Implications for human carcinomas and their treatments are discussed.

Inhibition of OXPHOS induces metabolic rewiring and reduces hypoxia in murine tumor models

IntroductionTumor hypoxia is a feature of many solid malignancies and is known to cause radio resistance. In recent years it has become clear that hypoxic tumor regions also foster an immunosuppressive phenotype and are involved in immunotherapy resistance. It has been proposed that reducing the tumors’ oxygen consumption will result in an increased oxygen concentration in the tissue and improve radio- and immunotherapy efficacy. The aim of this study is to investigate the metabolic rewiring of cancer cells by pharmacological attenuation of oxidative phosphorylation (OXPHOS) and subsequently reduce tumor hypoxia. Material and methodsThe metabolic effects of three OXPHOS inhibitors IACS-010759, atovaquone and metformin were explored by measuring oxygen consumption rate, extra cellular acidification rate, and [18F]FDG uptake in 2D and 3D cell culture. Tumor cell growth in 2D cell culture and hypoxia in 3D cell culture were analyzed by live cell imaging. Tumor hypoxia and [18F]FDG uptake in vivo following treatment with IACS-010759 was determined by immunohistochemistry and ex vivo biodistribution respectively. ResultsIn vitro experiments show that tumor cell metabolism is heterogeneous between different models. Upon OXPHOS inhibition, metabolism shifts from oxygen consumption through OXPHOS towards glycolysis, indicated by increased acidification and glucose uptake. Inhibition of OXPHOS by IACS-010759 treatment reduced diffusion limited tumor hypoxia in both 3D cell culture and in vivo. Although immune cell presence was lower in hypoxic areas compared with normoxic areas, it is not altered following short term OXPHOS inhibition. DiscussionThese results show that inhibition of OXPHOS causes a metabolic shift from OXPHOS towards increased glycolysis in 2D and 3D cell culture. Moreover, inhibition of OXPHOS reduces diffusion limited hypoxia in 3D cell culture and murine tumor models. Reduced hypoxia by OXPHOS inhibition might enhance therapy efficacy in future studies. However, caution is warranted as systemic metabolic rewiring can cause adverse effects.

ExoSloNano: Multi-Modal Nanogold Tags for identification of Macromolecules in Live Cells & Cryo-Electron Tomograms.

In situ cryo-Electron Microscopy (cryo-EM) enables the direct interrogation of structure-function relationships by resolving macromolecular structures in their native cellular environment. Tremendous progress in sample preparation, imaging and data processing over the past decade has contributed to the identification and determination of large biomolecular complexes. However, the majority of proteins are of a size that still eludes identification in cellular cryo-EM data, and most proteins exist in low copy numbers. Therefore, novel tools are needed for cryo-EM to identify the vast majority of macromolecules across multiple size scales (from microns to nanometers). Here, we introduce and validate novel nanogold probes that enable the detection of specific proteins using cryo-ET (cryo-Electron Tomography) and resin-embedded correlated light and electron microscopy (CLEM). We demonstrate that these nanogold probes can be introduced into live cells, in a manner that preserves intact molecular networks and cell viability. We use this system to identify both cytoplasmic and nuclear proteins by room temperature EM, and resolve associated structures by cryo-ET. We further employ gold particles of different sizes to enable future multiplexed labeling and structural analysis. By providing high efficiency protein labeling in live cells and molecular specificity within cryo-ET tomograms, we establish a broadly enabling tool that significantly expands the proteome available to electron microscopy.

Bioluminescence Resonance Energy Transfer (BRET) Assay to Measure Gq Recruitment to the Ghrelin Receptor.

This protocol chapter outlines a guide for investigating ghrelin receptor activation of Gq signaling using Bioluminescence Resonance Energy Transfer (BRET). Focusing on the eBRET2 and NanoBRET™ variants, the chapter covers steps from cell culture to transfection, ligand stimulation, and BRET measurements, offering a robust protocol to examine the temporal aspects of Gq signaling in live cells. This methodology facilitates a nuanced understanding of Gq-mediated cellular responses, but also provides researchers with a valuable tool for dissecting other related signaling pathways.

Measuring IP3 Generation in Real-Time Using a NanoBiT Luminescence Biosensor.

G protein-coupled receptors that activate Gq/11 regulate a range of physiological processes including neurotransmission, energy homeostasis, blood pressure regulation, and calcium homeostasis. Activation of Gq/11-coupled receptors stimulates the generation of inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular calcium release from the endoplasmic reticulum. This chapter describes an assay that uses a NanoBiT-IP3 luminescent biosensor to detect increases in IP3 in live cells. It describes how to perform these assays to assess signaling by the ghrelin receptor and the calcium-sensing receptor in HEK293 cells.

Measuring GPCR-Induced Intracellular Calcium Signaling Using a Quantitative High-Throughput Assay.

Alterations in intracellular calcium are integral to signal transduction pathways for many G-protein-coupled receptors, but this signaling is not well studied. This is mostly due to a lack of reliable, robust, high-throughput, quantitative methods to monitor intracellular calcium concentrations in live cells. Recently, we developed a reliable, robust, quantitative method to measure intracellular calcium levels in which HEK293 cell suspensions loaded with Fura-2/AM are placed in 96-well plates. Minimum and maximum intracellular calcium levels, which are required for converting fluorescence into calcium concentrations, are calibrated using EGTA to chelate calcium and ionomycin to load calcium into cells, respectively. Fluorescence is monitored with a PHERAstar FS plate reader. We provide a detailed method for this high-throughput assay that can be used to quantitate intracellular calcium in endogenous and exogenously (stable or transient) expressed GPCRs in HEK293 cells.

Vitamin U alleviates AFB1-induced hepatotoxicity in pregnant and lactating mice by regulating the Nrf2/Hmox1 pathway

This study investigated the protective effect of Vitamin U on liver injury induced by aflatoxin B1 (AFB1) in maternal mice. 25 pregnant ICR mice were randomly divided into five groups: the AFB1 group (AF, 0.3 mg AFB1/kg b.w.), the Vitamin U group (U, 50 mg Vitamin U/kg b.w.), the AFB1 + Vitamin U group (AU, 50 mg Vitamin U /kg b.w. + 0.3 mg AFB1/kg b.w.), the control group (DMSO), and the MOCK group (distilled water). They were administered substances by gavage every day for 28 days. Results indicated that exposure to AFB1 increased the liver index and caused histological disruptions. Elevated serum levels of ALT and ALP were observed, along with a significant increase in liver MDA content and a decrease in GSH-Px and T-SOD levels. Moreover, the Keap1 and Hmox1 gene was downregulated with statistical significance, while the IL1β and TNFα gene were significantly upregulated. Vitamin U was demonstrated by the organized structure of liver cells in tissue slices, effectively reducing liver cell necrosis. This intervention was associated with a significant decrease in serum ALT and ALP activities, as well as a significant decrease in liver MDA content. Additionally, there were significant increases in liver T-SOD and GSH-Px levels, along with upregulation of mRNA and protein expression of Nfr2, Hmox1 and Keap1, and downregulation of mRNA expression of the IL1β gene. In summary, Vitamin U mitigated oxidative stress-induced liver injury by modulating the Nrf2/Hmox1 signaling pathway and inflammatory factors affected by AFB1.

Novel dual-color fluorescence probe for monitoring the fluctuation of lysosomal polarity and viscosity during apoptosis

Apoptosis is pivotal in the pathogenesis of cancer and various other diseases. Real-time monitoring of apoptosis is crucial for assessing cell viability and facilitating drug screening. The process of apoptosis tends to cause changes in cellular microenvironmental factors. The variations of the microenvironment can serve as an indicator of the degree of apoptosis. Thus, the development of a smart and facile probe responding to cellular microenvironmental factors is significant for visualizing the apoptotic process. In this research, we designed a novel dual-color fluorescent probe, Lyso-DCPV, which incorporates N, N-diethyl coumarin as its polarity-responsive module, and the twisting double bond between N, N-diethyl coumarin and N-propyl quinoline as its viscosity-responsive module. The green fluorescence of Lyso-DCPV is sensitive to polarity changes, diminishing as polarity increases. Conversely, the red fluorescence intensifies with escalating viscosity. The probe specifically targets lysosomes, producing green fluorescence in live cells. Due to Lyso-DCPV’s acute sensitivity to changes in polarity and viscosity, it is well-suited to observe variations in the intracellular lysosomal microenvironment during drug-induced apoptosis. We also employed Lyso-DCPV for the dynamic assessment of polarity and viscosity during peroxide-induced apoptosis, enabling the observation of cell viability through variations in the probe’s emission color and intensity. The distinct probe will pave the way for novel strategies in evaluating apoptosis-related drugs. Moreover, Lyso-DCPV stands as an exemplary molecular instrument to elucidate the interplay between polarity and viscosity, facilitating the exploration of the interrelation in the cellular microenvironment during apoptosis.

Innate immune regulation in inflammation resolution and liver regeneration in drug-induced liver injury.

Drug-induced liver injury (DILI) is an acute liver injury that poses a significant threat to human health. In severe cases, it can progress into chronic DILI or even lead to liver failure. DILI is typically caused by either intrinsic hepatotoxicity or idiosyncratic metabolic or immune responses. In addition to the direct damage drugs inflict on hepatocytes, the immune responses and liver inflammation triggered by hepatocyte death can further exacerbate DILI. Initially, we briefly discussed the differences in immune cell activation based on the type of liver cell death (hepatocytes, cholangiocytes, and LSECs). We then focused on the role of various immune cells (including macrophages, monocytes, neutrophils, dendritic cells, liver sinusoidal endothelial cells, eosinophils, natural killer cells, and natural killer T cells) in both the liver injury and liver regeneration stages of DILI. This article primarily reviews the role of innate immune regulation mediated by these immune cells in resolving inflammation and promoting liver regeneration during DILI, as well as therapeutic approaches targeting these immune cells for the treatment of DILI. Finally, we discussed the activation and function of liver progenitor cells (LPCs) during APAP-induced massive hepatic necrosis and the involvement of chronic inflammation in DILI.

Wavelet‐Forward Family Enabling Stitching‐Free Full‐Field Fourier Ptychographic Microscopy

AbstractFourier ptychographic microscopy (FPM) breaks through the resolution limitations of conventional optical systems, which offer a full‐field view and high resolution without additional mechanical scanning. However, conventional image‐domain optimizations require trade‐offs between correction efficacy, data redundancy, and reconstruction accuracy. Furthermore, the existing linear time‐invariant model for actual nonlinear, time‐varying optical systems leads to forward model mismatch, complicating the corrections of the vignetting effect. To overcome these challenges and achieve stitching‐free FPM, a family of forward wavelet‐transform models (WL‐FPM) is proposed. WL‐FPM employs the reversibility of the wavelet transform for high‐fidelity reconstruction in the multiscale feature domain. The wavelet loss function is updated in each iteration, and non‐convex optimization is solved by complex back diffraction. WL‐FPM offers stitching‐free, high‐resolution, and robust reconstruction under various challenging conditions, including vignetting effects, LED position mismatch, intensity fluctuations, and high‐level noise environments, which outperform conventional FPM methods. Under a 4X objective with NA 0.1, WL‐FPM achieves a 435‐nm resolution and stitching‐free full‐field reconstruction of a 3.328 × 3.328 mm2 pathological section with distinct subcellular organelles. In live cell imaging, it provides a full‐field observation with distinct lipids in a single cell. A large number of simulation and experimental results demonstrate its potential for biomedical applications.

CRISPR/Cas13 sgRNA-Mediated RNA-RNA Interaction Mapping in Live Cells with APOBEC RNA Editing.

Current research on long non-coding RNA (lncRNA) has predominantly focused on identifying their protein partners and genomic binding sites, leaving their RNA partners largely unknown. To address this gap, the study has developed a method called sarID (sgRNA scaffold assisted RNA-RNA interaction detection), which integrates Cas13-based RNA targeting, sgRNA engineering, and proximity RNA editing to investigate lncRNA-RNA interactomes. By applying sarID to the lncRNA NEAT1, over one thousand previously unidentified binding transcripts are discovered. sarID is further expanded to investigate binders of XIST, MALAT1, NBR2, and DANCR, demonstrating its broad applicability in identifying lncRNA-RNA interactions. The findings suggest that lncRNAs may regulate gene expression by interacting with mRNAs, expanding their roles beyond known functions as protein scaffolds, miRNA sponges, or guides for epigenetic modulators. sarID has the potential to be adapted for studying other specific RNAs, providing a novel immunoprecipitation-free method for uncovering RNA partners and facilitating the exploration of the RNA-RNA interactome.

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells.

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describea split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

The endoplasmic reticulum as an active liquid network

The peripheral endoplasmic reticulum (ER) forms a dense, interconnected, and constantly evolving network of membrane-bound tubules in eukaryotic cells. While individual structural elements and the morphogens that stabilize them have been described, a quantitative understanding of the dynamic large-scale network topology remains elusive. We develop a physical model of the ER as an active liquid network, governed by a balance of tension-driven shrinking and new tubule growth. This minimalist model gives rise to steady-state network structures with density and rearrangement timescales predicted from the junction mobility and tubule spawning rate. Several parameter-independent geometric features of the liquid network model are shown to be representative of ER architecture in live mammalian cells. The liquid network model connects the timescales of distinct dynamic features such as ring closure and new tubule growth in the ER. Furthermore, it demonstrates how the steady-state network morphology on a cellular scale arises from the balance of microscopic dynamic rearrangements.

Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes.

Rhoifolin (ROF) exhibits a diverse range of biological activities, encompassing anticancer, hepatoprotective, antidiabetic, antirheumatic, and antiviral properties. However, the specific protective effects and possible mechanisms of the compound against T-cell-mediated autoimmune hepatitis have not been previously elucidated. In the present study, adult male mice were administered Con A (20 mg/kg, intravenously) for 8 h. In the treated groups, mice were pretreated with ROF daily (20 mg/kg and 40 mg/kg, orally) for 7 days before Con A intoxication. The results showed that ROF significantly decreased serum biochemical indices (ALT, AST, ALP, and LDH) and regulated related oxidative stress indicators (MDA, SOD, and GSH), reduced hepatic necrosis areas and immune cells infiltration, inhibited the release of various inflammatory factors (TNF-α, IFN-γ, IL-2, and IL-17), and improved hepatic tissue apoptosis, thereby alleviating hepatic damage induced by Con A. Additionally, we have also confirmed that ROF efficiently inhibited Th1/Th17 cells polarization via modulation of the JAK2/JAK3/STAT1/STAT3 signaling pathways both in vivo and in vitro. Moreover, the molecular mechanism examination also demonstrated that ROF regulated apoptotic cascade signaling through IL-6/JAK2/STAT1/STAT3 controlling BNIP3 activity in primary hepatocytes. These effects were in good agreement with the bioinformatics analysis of ROF treatment for AIH. In conclusion, our findings provide new insights into the potential use of ROF for AIH therapy, which may result from the specific regulation of the T cell subtype polarization and the apoptosis of liver cells via modulation of the JAKs/STATs signaling pathways.

Pyrene Based Schiff Base for Cascade Fluorescent "On-Off" Detection of Aluminium(III) and Fluoride ions and its Applications in Live Cells Imaging.

An easy-to-prepare pyrene-based Schiff base PNZ was synthesized by condensing equimolar amount of 1-pyrenebutyric hydrazide with 2-hydroxy-naphthaldehyde, and employed as a fluorescent chemosensor for in-situ cascade detection of aluminium (Al3+) and fluoride (F-)ions. In DMSO:H2O (1:1, v/v), the weakly emissive PNZ showed a significant fluorescence enhancement at 455 nm selectively upon the addition of Al3+ due to the complexation-induced formation of a pyrene excimer. Schiff base PNZ and Al3+ formed a complex in 2:1 binding ratio with the estimated binding constants of K1:1 = 9826.01 M-1 and K2:1 = 3188.49 M-1. The sensing mechanism was explored by performing quantum mechanical calculations and 1H NMR titration of PNZ with Al3+. The in-situ formed PNZ-Al3+ complex species enabled the fluorescent turn-off detection of F-. Using PNZ and PNZ-Al3+, the concentrations of Al3+ and F- ions can be detected down to 2.89×10-7 M and 1.88×10-7 M, respectively. The cytotoxicity of the PNZ and its ability to bioimage Al3+ and F- ions was examined in the human cervical cancer cell line. Finally, the receptor PNZ was applied for the quantification of Al3+ and F- ions in various real samples, such as tap water, river water, rainwater, mouthwash, and toothpaste.

Quantitative dynamics of intracellular NMN by genetically encoded biosensor

Nicotinamide mononucleotide (NMN) is the direct precursor and a major booster of NAD+ with increasing applications in NAD+- and aging-related pathologies. However, measuring live cell NMN dynamics was not possible, leaving key questions in NMN uptake and intracellular regulation unanswered. Here we developed genetically encoded bioluminescent and fluorescent sensors to quantify subcellular NMN in live cells by engineering specific NMN-responsive protein scaffolds fused to luciferase and fluorescent proteins. The sensor dissected the multimechanistic uptake of exogenous NMN and nicotinamide riboside (NR) in live cells and further measured the NMN levels across different subcellular compartments, as well as the perturbed NMN/NAD+ ratios by external supplements. Moreover, we measured the NMN regulation by NAD(H) hydrolase Nudts and peroxisomal carrier Pxmp2 and identified Slc25a45 as a potential mitochondrial NMN regulator for its unique fingerprint on the local NMN/NAD+ ratio. Collectively, the genetically encoded sensors provide a useful tool for visualizing NMN metabolism.

Novel Far-Red Fluorescent 1,4-Dihydropyridines for L-Type Calcium Channel Imaging.

Upregulation of L-type calcium channels (LTCCs) is implicated in a range of cardiovascular and neurological disorders. Therefore, the development of toolboxes that unlock fast imaging protocols in live cells is coveted. Herein, we report a library of first-in-class far-red small-molecule-based fluorescent ligands (FluoDiPines), able to target LTCCs. All fluorescent ligands were evaluated in whole-cell patch-clamp and live-cell Ca2+ imaging whereby FluoDiPine 6 was found to be the best candidate for live-cell fluorescence imaging. Low concentration of FluoDiPine 6 (50 nM) and a quick labeling protocol (5 min) are successfully applied to fixed and live cells to image LTCCs with good specificity.