Live-cell Fluorescence Imaging Research Articles

Fluorogenic Rhodamine Probes with Pyrrole Substitution Enables STED and Lifetime Imaging of Lysosomes in Live Cells.

Fluorogenic dyes with high brightness, large turn-on ratios, excellent photostability, favorable specificity, low cytotoxicity, and high membrane permeability are essential for high-resolution fluorescence imaging in live cells. In this study, we endowed these desirable properties to a rhodamine derivative by simply replacing the N, N-diethyl group with a pyrrole substituent. The resulting dye, Rh-NH, exhibited doubled Stokes shifts (54 nm) and a red-shift of more than 50 nm in fluorescence spectra compared to Rhodamine B. Rh-NH preferentially exists in a non-emissive but highly permeable spirolactone form. Upon binding to lysosomes, the collective effects of low pH, low polarity, and high viscosity endow Rh-NH with significant fluorescence turn-on, making it a suitable candidate for wash-free, high-contrast lysosome tracking. Consequently, Rh-NH enabled us to successfully explore stimulated emission depletion (STED) super-resolution imaging of lysosome dynamics, as well as fluorescence lifetime imaging of lysosomes in live cells.

Delivery of Fluorescent Nanoprobes into the Cytoplasm Using Cell-Penetrating Peptides for Live-Cell Super-Resolution Fluorescence Imaging

Delivery of Fluorescent Nanoprobes into the Cytoplasm Using Cell-Penetrating Peptides for Live-Cell Super-Resolution Fluorescence Imaging

Characterization of a nuclear transport factor 2-like domain-containing protein in Plasmodium berghei

BackgroundPlasmodium lacks an mRNA export receptor ortholog, such as yeast Mex67. Yeast Mex67 contains a nuclear transport factor 2 (NTF2)-like domain, suggesting that NTF2-like domain-containing proteins might be associated with mRNA export in Plasmodium. In this study, the relationship between mRNA export and an NTF2-like domain-containing protein, PBANKA_1019700, was investigated using the ANKA strain of rodent malaria parasite Plasmodium berghei.MethodsThe deletion mutant Δ1019700 was generated by introducing gene-targeting vectors into the P. berghei ANKA genome, and parasite growth and virulence were examined. To investigate whether PBANKA_1019700 is involved in mRNA export, live-cell fluorescence imaging and immunoprecipitation coupled to mass spectrometry (IP-MS) were performed using transgenic parasites expressing fusion proteins (1019700::mCherry).ResultsDeletion of PBANKA_1019700 affected the sexual phase but not the asexual phase of malaria parasites. Live-cell fluorescence imaging showed that PBANKA_1019700 localizes to the cytoplasm. Moreover, IP-MS analysis of 1019700::mCherry indicated that PBANKA_1019700 interacts with ubiquitin-related proteins but not nuclear proteins.ConclusionsPBANKA_1019700 is a noncanonical NTF2-like superfamily protein.

Infectious Virus Tracking by Fluorescent Live Cell Imaging in Primary Cells.

To successfully infect a cell, HIV-1 has to overcome several host barriers while exploiting cellular cofactors. HIV-1 infection is highly inefficient with the great majority of viral particles not being able to successfully integrate into the target cell genome. Nonproductive HIV-1 particles are degraded or accumulated in cellular compartments. Thus, it becomes hard to distinguish between viral behaviors that lead to effectively infecting the cell from the ones that do not by using traditional methods. Here, we describe the infectious virus tracking method that detects and quantifies individual fluorescent viral particles over time and links viral particle behavior to its infectivity. This method employs live-cell imaging at ultra-low MOIs to detect the outcome of infection for every HIV-1 particle.

Live-Cell Fluorescence Imaging for Virus-Host Interactions.

Recent technological advances in microscopy have facilitated novel approaches to investigate host-pathogen interactions. In particular, improvements in both microscope hardware and engineered biosensors have helped to overcome barriers to live-cell imaging with fluorescence microscopy. Live fluorescent microscopy allows for the detection of discrete signaling events and protein localization, improving our ability to assess the effects of pharmacologic agents, microbes, or infection with high temporal resolution. Here we describe a protocol for long-term live-cell fluorescence imaging of virus infected cell lines.

The Na/K-ATPase α1/Src Signaling Axis Regulates Mitochondrial Metabolic Function and Redox Signaling in Human iPSC-Derived Cardiomyocytes.

Na/K-ATPase (NKA)-mediated regulation of Src kinase, which involves defined amino acid sequences of the NKA α1 polypeptide, has emerged as a novel regulatory mechanism of mitochondrial function in metazoans. Mitochondrial metabolism ensures adequate myocardial performance and adaptation to physiological demand. It is also a critical cellular determinant of cardiac repair and remodeling. To assess the impact of the proposed NKA/Src regulatory axis on cardiac mitochondrial metabolic function, we used a gene targeting approach in human cardiac myocytes. Human induced pluripotent stem cells (hiPSC) expressing an Src-signaling null mutant (A420P) form of the NKA α1 polypeptide were generated using CRISPR/Cas9-mediated genome editing. Total cellular Na/K-ATPase activity remained unchanged in A420P compared to the wild type (WT) hiPSC, but baseline phosphorylation levels of Src and ERK1/2 were drastically reduced. Both WT and A420P mutant hiPSC readily differentiated into cardiac myocytes (iCM), as evidenced by marker gene expression, spontaneous cell contraction, and subcellular striations. Total NKA α1-3 protein expression was comparable in WT and A420P iCM. However, live cell metabolism assessed functionally by Seahorse extracellular flux analysis revealed significant reductions in both basal and maximal rates of mitochondrial respiration, spare respiratory capacity, ATP production, and coupling efficiency. A significant reduction in ROS production was detected by fluorescence imaging in live cells, and confirmed by decreased cellular protein carbonylation levels in A420P iCM. Taken together, these data provide genetic evidence for a role of NKA α1/Src in the tonic stimulation of basal mitochondrial metabolism and ROS production in human cardiac myocytes. This signaling axis in cardiac myocytes may provide a new approach to counteract mitochondrial dysfunction in cardiometabolic diseases.

Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy.

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of the mitochondrial network allows healthy mitochondria to meet cellular demands; however, the loss of this capacity has been related to the development or progression of different pathologies. In skeletal muscle, mitochondrial density and distribution changes are observed in physiological and pathological conditions such as exercise, aging, and obesity, among others. Therefore, the study of the mitochondrial network may provide a better understanding of mechanisms related to those conditions. Here, a protocol for mitochondria imaging of live-skeletal muscle fibers from rats is described. Fibers are manually dissected in a relaxing solution and incubated with a fluorescent live-cell imaging indicator of mitochondria (tetramethylrhodamine ethyl ester, TMRE). The mitochondria signal is recorded by confocal microscopy using the XYZ scan mode to obtain confocal images of the intermyofibrillar mitochondrial (IMF) network. After that, the confocal images are processed by thresholding and binarization. The binarized confocal image accounts for the positive pixels for mitochondria, which are then counted to obtain the mitochondrial density. The mitochondrial network in skeletal muscle is characterized by a high density of IMF population, which has a periodic longitudinal distribution similar to that of T-tubules (TT).The Fast Fourier Transform (FFT) is a standard analysis technique performed to evaluate the distribution of TT that allows finding the distribution frequency and the level of their organization. In this protocol, the implementation of the FFT algorithm is described for the analysis of the longitudinal mitochondrial distribution in skeletal muscle.

An antioxidant screen identifies ascorbic acid for prevention of light-induced mitotic prolongation in live cell imaging

Phototoxicity is an important issue in fluorescence live imaging of light-sensitive cellular processes such as mitosis. Among several approaches to reduce phototoxicity, the addition of antioxidants to the media has been used as a simple method. Here, we analyzed the impact of phototoxicity on the mitotic progression in fluorescence live imaging of human cells and performed a screen to identify the most efficient antioxidative agents that reduce it. Quantitative analysis shows that high amounts of light illumination cause various mitotic defects such as prolonged mitosis and delays of chromosome alignment and centrosome separation. Among several antioxidants, our screen reveals that ascorbic acid significantly alleviates these phototoxic effects in mitosis. Furthermore, we demonstrate that adding ascorbic acid to the media enables fluorescence imaging of mitotic events at very high temporal resolution without obvious photodamage. Thus, this study provides an optimal method to effectively reduce the phototoxic effects in fluorescence live cell imaging.

6-azido and 6-azidomethyl uracil nucleosides

Azido nucleosides have been utilized for click reactions, metabolic incorporation into cellular DNA, and fluorescent imaging of live cells. Two classes of 6-azido modified uracil nucleosides; one with azido group directly attached to uracil ring and second with azido group attached via methylene linker are described. The 6-azido-2’-deoxyuridine (6-AdU) was prepared in 55% overall yield by lithiation-based regioselective C6-iodination of silyl protected 2’-deoxyuridine followed by treatment with sodium azide and deprotection with TBAF. Lithiation-based C6-alkylation of the protected uridine with methyl iodide followed by the oxidation of the 6-methyl product with selenium dioxide and the subsequent mesylation and azidation of the resulting 6-hydroxymethyl group gave after deprotection 6-azidomethyluridine (6-AmU) in 61% overall yield. Direct lithiation-based C6-hydroxymethylation followed by mesylation/azidation sequence and deprotection provided 6-AmU or 6-azidomethyl-2’-deoxyuridine (6-AmdU). Yields for the lithiation-based regioselective C6-iodination and alkylation were higher for uridine than 2’-deoxyuridine derivatives and they appear to be less dependent on the sugar protection group used. Strain promoted click reactions of 6-AdU and 6-AmdU with symmetrically fused cyclopropyl cyclooctyne (OCT) provided fluorescent triazoles. DFT-calculated dihedral angles and energy differences for the favored anti and syn conformation of 6-AdU and 6-AmdU versus their C5 azido counterparts are discussed.

Mild Photothermal Therapy Prevents Posterior Capsule Opacification through Cytoskeletal Remodeling.

Cataract is the first leading cause of blindness in the world and posterior capsule opacification (PCO) is the most common long-term complication after surgery. The primary pathogenic processes contributing to PCO are the proliferation and migration of residual lens epithelial cells (LECs). This study aimed to explore the mild photothermal effect on LECs. Interestingly, this work finds that the mild photothermal effect significantly inhibited the proliferation and migration of LECs. The live cell fluorescence imaging reveals that the remodeling of the actin cytoskeleton and cell morphology attributed to the inhibition effect. Further mechanistic studies at molecular level suggest that the mild photothermal effect can regulate the phosphorylation of ERM, YAP, and Cofilin and thereby affect the proliferation and migration of LECs. In order to explore the potential clinical application of mild photothermal therapy for PCO prevention, PDA/PVA gel rings with photothermal effect is prepared by the repeated freeze-thaw method and conducted experiments in vivo, which achieved favorable PCO prevention effect. Overall, this study shows that the mild photothermal effect can regulate the proliferation and migration of LECs through cytoskeletal remodeling and the results of experiments in vivo demonstrate that mild photothermal effect is a promising approach for PCO prevention.

Large Stokes shift fluorescent RNAs for dual-emission fluorescence and bioluminescence imaging in live cells.

Fluorescent RNAs, aptamers that bind and activate small fluorogenic dyes, have provided a particularly attractive approach to visualizing RNAs in live cells. However, the simultaneous imaging of multiple RNAs remains challenging due to a lack of bright and stable fluorescent RNAs with bio-orthogonality and suitable spectral properties. Here, we develop the Clivias, a series of small, monomeric and stable orange-to-red fluorescent RNAs with large Stokes shifts of up to 108 nm, enabling the simple and robust imaging of RNA with minimal perturbation of the target RNA's localization and functionality. In combination with Pepper fluorescent RNAs, the Clivias enable the single-excitation two-emission dual-color imaging of cellular RNAs and genomic loci. Clivias can also be used to detect RNA-protein interactions by bioluminescent imaging both in live cells and in vivo. We believe that these large Stokes shift fluorescent RNAs will be useful tools for the tracking and quantification of multiple RNAs in diverse biological processes.

Plasmodium ARK2 and EB1 drive unconventional spindle dynamics, during chromosome segregation in sexual transmission stages

The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei, using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite.

RNA-Selective Small Molecule Ligands: The Recent Advances in Live Cell Imaging and Drug Discovery.

RNA structures, including those formed from coding and noncoding RNAs, alternative to protein-based drug targets, could be a promising target of small molecules for drug discovery against various human diseases, particularly in anticancer, antibacterial and antivirus development. The normal cellular activity of cells is critically dependent on the function of various RNA molecules generated from DNA transcription. Moreover, many studies support that mRNA-targeting small molecules may regulate the synthesis of disease-related proteins via the non-covalent mRNA-ligand interactions that do not involve gene modification. RNA-ligand interaction is thus an attractive approach to address the challenge of "undruggable" proteins in drug discovery because the intracellular activity of these proteins is hard to be suppressed with small molecule ligands. We selectively surveyed a specific area of RNA structure-selective small molecule ligands in fluorescence live cell imaging and drug discovery because the area was currently underexplored. This state-of-the-art review thus mainly focuses on the research published within the past three years and aims to provide the most recent information on this research area; hopefully, it could be complementary to the previously reported reviews and give new insights into the future development on RNA-specific small molecule ligands for live cell imaging and drug discovery.

Liposome Based Near-Infrared Sensors for the Selective Detection of Hydrogen Sulfide.

Cyanine dye-based new amphiphilic compound NIR-Amp has been synthesised. NIR-Amp was embedded with phospholipids DOPC and DPPC to form liposomes based nanoscale chemical sensors NIR-Lip1 and NIR-Lip2. Here, two different phospholipids were used to demonstrate the influence of lipid structure, composition and fluidity on sensing of nanosensors. Both the probes show NIR absorption maximum at 790 nm and emission maximum at 815 nm. H2 S-triggered thiolation resulted a remarkable change in color from green to pale yellow. A decrease in UV-Vis absorption and emission in the NIR region was observed only with H2 S. NIR-Lip1 and NIR-Lip2 are highly selective for H2 S with a LOD of 0.57 μM and 1.24 μM, respectively. It was observed that in a solid-like gel state, NIR-Lip1 is slightly more sensitive towards H2 S than fluid-like NIR-Lip2. The H2 S sensing mechanism was confirmed by ESI-mass and infrared (IR) spectroscopic analysis. Based on the high sensitivity and selectivity, NIR-Lip1 was employed to detect H2 S in vegetable samples. Further, the probes are found to be non-toxic and established for H2 S fluorescence imaging in live cells.

Accessing a Long‐Fluorescence‐Lifetime BODIPY Dye via Efficient Förster Resonance Energy Transfer Induced by Host‐Guest Doping

AbstractThe development of pure organic room temperature phosphorescence (RTP) luminophores with high quantum yield and long emission lifetime has attracted considerable attention due to their extensive optoelectronic and biomedical applications. The commonly used BODIPY dyes have strong UV‐Vis absorption, a relatively sharp emission peak, high quantum yield, and a short emission lifetime. By expending the molecular doping approach, we herein successfully achieved a long‐fluorescence‐lifetime BODIPY dye via efficient Förster resonance energy transfer (FRET) from RTP molecule o‐AI‐Cz to the short‐lived fluorescence emitter Bodipy‐2I. This host‐guest material possessed an ultralong lifetime of up to 595 ms and a long emission wavelength under ambient conditions, which has great potential in data encryption and anti‐counterfeiting applications. Moreover, this easily prepared FRET system exhibited an efficient response to two‐photon excitation in living cell fluorescence imaging.

From the membrane to the nucleus: mechanical signals and transcription regulation.

Mechanical forces drive and modulate a wide variety of processes in eukaryotic cells including those occurring in the nucleus. Relevantly, forces are fundamental during development since they guide lineage specifications of embryonic stem cells. A sophisticated macromolecular machinery transduces mechanical stimuli received at the cell surface into a biochemical output; a key component in this mechanical communication is the cytoskeleton, a complex network of biofilaments in constant remodeling that links the cell membrane to the nuclear envelope. Recent evidence highlights that forces transmitted through the cytoskeleton directly affect the organization of chromatin and the accessibility of transcription-related molecules to their targets in the DNA. Consequently, mechanical forces can directly modulate transcription and change gene expression programs. Here, we will revise the biophysical toolbox involved in the mechanical communication with the cell nucleus and discuss how mechanical forces impact on the organization of this organelle and more specifically, on transcription. We will also discuss how live-cell fluorescence imaging is producing exquisite information to understand the mechanical response of cells and to quantify the landscape of interactions of transcription factors with chromatin in embryonic stem cells. These studies are building new biophysical insights that could be fundamental to achieve the goal of manipulating forces to guide cell differentiation in culture systems.

Backscatter from therapeutic doses of ionizing irradiation does not impair cell migration on titanium implants in vitro

ObjectiveThe influence of radiation backscatter from titanium on DNA damage and migration capacity of human osteoblasts (OBs) and mesenchymal stem cells (MSCs) may be critical for the osseointegration of dental implants placed prior to radiotherapy. In order to evaluate effects of radiation backscatter, the immediate DNA damage and migration capacity of OBs and MSCs cultured on titanium or plastic were compared after exposure to ionizing irradiation. Materials and methodsHuman OBs and MSCs were seeded on machined titanium, moderately rough fluoride-modified titanium, or tissue culture polystyrene, and irradiated with nominal doses of 2, 6, 10, or 14 Gy. Comet assay was performed immediately after irradiation, while a scratch wound healing assay was initiated 24 h post-irradiation. Fluorescent live cell imaging documented the migration.ResultsDNA damage increased with higher dose and with backscatter from titanium, and MSCs were significantly more affected than OBs. All doses of radiation accelerated the cell migration on plastic, while only the highest dose of 10 Gy inhibited the migration of both cell types on titanium.ConclusionsHigh doses (10 Gy) of radiation inhibited the migration capacity of both cell types on titanium, whereas lower doses (2 and 6 Gy) did not affect the migration of either OBs or MSCs.Clinical relevanceFractionated doses of 2 Gy/day, as distributed in conventional radiotherapy, appear not to cause severe DNA damage or disturb the migration of OBs or MSCs during osseointegration of dental implants.

Altered TRPM7-Dependent Calcium Influx in Natural Killer Cells of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disabling multisystemic condition. The pathomechanism of ME/CFS remains unestablished; however, impaired natural killer (NK) cell cytotoxicity is a consistent feature of this condition. Calcium (Ca2+) is crucial for NK cell effector functions. Growing research recognises Ca2+ signalling dysregulation in ME/CFS patients and implicates transient receptor potential ion channel dysfunction. TRPM7 (melastatin) was recently considered in the pathoaetiology of ME/CFS as it participates in several Ca2+-dependent processes that are central to NK cell cytotoxicity which may be compromised in ME/CFS. TRPM7-dependent Ca2+ influx was assessed in NK cells isolated from n = 9 ME/CFS patients and n = 9 age- and sex-matched healthy controls (HCs) using live cell fluorescent imaging techniques. Slope (p < 0.05) was significantly reduced in ME/CFS patients compared with HCs following TRPM7 activation. Half-time of maximal response (p < 0.05) and amplitude (p < 0.001) were significantly reduced in the HCs compared with the ME/CFS patients following TRPM7 desensitisation. Findings from this investigation suggest that TRPM7-dependent Ca2+ influx is reduced with agonism and increased with antagonism in ME/CFS patients relative to the age- and sex-matched HCs. The outcomes reported here potentially reflect TRPM3 dysfunction identified in this condition suggesting that ME/CFS is a TRP ion channelopathy.

1653-P: Brown Adipocyte–Secreted Factors for Treating Type 1 Diabetes Mellitus

Hyperglycemia during type 1 Diabetes (T1D) is driven not only by a lack of insulin secretion due to autoimmune destruction of the pancreatic β-cells, but also by hypersecretion of glucagon from pancreatic α-cells. Insulin therapy can replace the lack of insulin secretion, but there is no effective therapeutic tool to counter the hyperglucagonemia. Brown adipose tissue (BAT) has gained attention for treating metabolic diseases due to its role as an endocrine organ and its effect on whole-body energy metabolism. Subcutaneous transplantation of embryonic BAT into T1D mouse models reverses hyperglycemia, independent of insulin but accompanied by reduced plasma glucagon levels, which suggests that lowering plasma glucagon plays a central role in restoring euglycemia. However, the molecular mechanism(s) underlying this action remain unclear. Towards discovering these mechanisms, we have examined the effects of secretions from immortalized brown adipocytes (imBAT) on pancreatic α-cells and mouse models of T1D. We show that the large molecule fraction (&amp;gt;100kDa) of imBAT-secreted factors suppresses glucagon secretion from both mouse and human pancreatic α-cells. Live cell fluorescence imaging shows that this fraction inhibits both intracellular Ca2+ and cAMP activities, and these inhibitions are ameliorated by blocking the insulin receptor, suggesting that imBAT-secreted factors act, at least in part, through these receptors. Injecting this imBAT-secreted fraction into the NOD mouse model reverses hyperglycemia (242 ± 47.4 to 146 ± 24.7mg/dL, n=8), suppresses hyperglucagonemia (77.6 ± 55.4 to 10.7 ± 4.75pg/mL, n=8), improves glucose tolerance (n=3), and increases thermogenic activity and differentiation of endogenous BAT (n=7). Our data provide insights into the mechanisms of long-term recovery of euglycemia by BAT transplants, and further fractionation experiments to isolate active factors are ongoing. Disclosure J.Lee: None. D.W.Piston: None. Funding The Leona M. and Harry B. Helmsley Charitable Trust (2305-06050); National Institutes of Health (R01DK123301, P30DK020579)

Development of a fluorescent probe based on a tricyano structure for the detection of PhSH in environmental and biological samples

In this study, a NIR fluorescent probe based on ICT principles was developed for the detection of phenylthiophenol. An excellent fluorescent mother nucleus is constructed with tricyano groups, and benzenesulfonate was introduced as a specific recognition site for thiophene, which can be used for rapid detection of thiophenol. The probe has a significant Stokes shift (220 nm). Meanwhile, it had rapid response to thiophene and high specificity. The fluorescence intensity of the probe at 700 nm showed a good linear relationship with thiophene concentration in the range of 0 to 100 μM, and the detection limit was as low as 45 nM. The probe had also been successfully applied to the detection of thiophene in real water samples. MTT assay showed low cytotoxicity and excellent fluorescence imaging in live cells.