Live-cell Fluorescence Imaging Research Articles

Cell Imaging: Photoactive Hybrid AuNR‐Pt@Ag2S Core–Satellite Nanostructures for Near‐Infrared Quantitive Cell Imaging (Adv. Funct. Mater. 46/2017)

A photoactive hybrid nanostructure for near-infrared quantitative cell imaging is reported by Hua Kuang and co-workers in article number 1703408. AuNanorod-PtAgS nanostructures are obtained with high yield. Application of the composite in fluorescent live cell imaging reveals a limit of detection for microRNAs of 0.0082 attomol per ng RNA. This system could provide further inside into the fate of microRNA in live cells.

Highly Efficient Visible Blue-Emitting Black Phosphorus Quantum Dot: Mussel-Inspired Surface Functionalization for Bioapplications.

The preparation of blue-emitting black phosphorus quantum dots (BPQDs) is based on the liquid-phase exfoliation of bulk BP. We report the synthesis of soluble BPQDs showing a strong visible blue-light emission. Highly fluorescent (photoluminescence quantum yield of ≈5% with the maximum emission (λmax) at ≈437 nm) and dispersible BPQDs in various organic solvents are first prepared by simple ultrasonication of BP crystals in chloroform in the ambient atmosphere. Furthermore, simple mussel-inspired surface functionalization of BPQDs with catechol-grafted poly(ethylene glycol) in basic buffer afforded water-soluble blue-emitting BPQDs showing long-term fluorescence stability, very low cytotoxicity, and excellent fluorescence live cell imaging capability.

Green synthesis of lignin based fluorescent nanocolorants for live cell imaging

In this study, lignin has been used as stabilizing agent during electrochemical synthesis of copper based nanocolorants. Brown, green and blue colored colloidal suspensions of nano particles have been produced under different electrolysis process conditions using copper electrodes. Average particle sizes are found to be 11 nm, 50 nm and 151 nm for green, blue and brown, respectively. Nanocolorants exhibit fluorescent properties under blue, green and UV filters and possess good antibacterial efficiency (9–12 mm zone of inhibition). They are nontoxic and efficient for live cell fluorescent imaging with L929 cells. Cells stained with green nanocolorant show highest fluorescent intensity among all synthesized colorants and pure lignin.

ALS-associated mutation SOD1G93A leads to abnormal mitochondrial dynamics in osteocytes

While the death of motor neuron is a pathological hallmark of amyotrophic lateral sclerosis (ALS), defects in other cell types or organs may also actively contribute to ALS disease progression. ALS patients experience progressive skeletal muscle wasting that may not only exacerbate neuronal degeneration, but likely has a significant impact on bone function. In our previous published study, we have discovered severe bone loss in an ALS mouse model with overexpression of ALS-associated mutation SOD1G93A (G93A). Here we further provide a mechanistic understanding of the bone loss in ALS animal and cellular models. Combining mitochondrial fluorescent indicators and confocal live cell imaging, we discovered abnormalities in mitochondrial network and dynamics in primary osteocytes derived from the same ALS mouse model G93A. Those mitochondrial defects occur in ALS mice after the onset of neuromuscular symptoms, indicating that mitochondria in bone cells respond to muscle atrophy during ALS disease progression. To examine whether ALS mutation has a direct contribution to mitochondrial dysfunction independent of muscle atrophy, we evaluated mitochondrial morphology and motility in cultured osteocytes (MLO-Y4) with overexpression of mitochondrial targeted SOD1G93A. Compared with osteocytes overexpressing the wild type SOD1 as a control, the SOD1G93A osteocytes showed similar defects in mitochondrial network and dynamic as that of the primary osteocytes derived from the ALS mouse model. In addition, we further discovered that overexpression of SOD1G93A enhanced the expression level of dynamin-related protein 1 (Drp1), a key protein promoting mitochondrial fission activity, and reduced the expression level of optic atrophy protein 1 (OPA1), a key protein related to mitochondrial fusion. A specific mitochondrial fission inhibitor (Mdivi-1) partially reversed the effect of SOD1G93A on mitochondrial network and dynamics, indicating that SOD1G93A likely promotes mitochondrial fission, but suppresses the fusion activity. Our data provide the first evidence that mitochondria show abnormality in osteocytes derived from an ALS mouse model. The accumulation of mutant SOD1G93A protein inside mitochondria directly causes dysfunction in mitochondrial dynamics in cultured MLO-Y4 osteocytes. In addition, the ALS mutation SOD1G93A-mediated dysfunction in mitochondrial dynamics is associated with an enhanced apoptosis in osteocytes, which could be a potential mechanism underlying the bone loss during ALS progression.

PH-Dependant Antifungal Activity of Valproic Acid against the Human Fungal Pathogen Candida albicans.

Current antifungal drugs suffer from limitations including toxicity, the emergence of resistance and decreased efficacy at low pH that are typical of human vaginal surfaces. Here, we have shown that the antipsychotic drug valproic acid (VPA) exhibited a strong antifungal activity against both sensitive and resistant Candida albicans in pH condition similar to that encountered in vagina. VPA exerted a strong anti-biofilm activity and attenuated damage of vaginal epithelial cells caused by C. albicans. We also showed that VPA synergizes with the allylamine antifungal, Terbinafine. We undertook a chemogenetic screen to delineate biological processes that underlies VPA-sensitivity in C. albicans and found that vacuole-related genes were required to tolerate VPA. Confocal fluorescence live-cell imaging revealed that VPA alters vacuole integrity and support a model where alteration of vacuoles contributes to the antifungal activity. Taken together, this study suggests that VPA could be used as an effective antifungal against vulvovaginal candidiasis.

Dissecting the Native Architecture and Dynamics of Cyanobacterial Photosynthetic Machinery

The structural dynamics and flexibility of cell membranes play fundamental roles in the functions of the cells, i.e., signaling, energy transduction, and physiological adaptation. The cyanobacterial thylakoid membrane represents a model membrane that can conduct both oxygenic photosynthesis and respiration simultaneously. In this study, we conducted direct visualization of the global organization and mobility of photosynthetic complexes in thylakoid membranes from a model cyanobacterium, Synechococcus elongatus PCC 7942, using high-resolution atomic force, confocal, and total internal reflection fluorescence microscopy. We visualized the native arrangement and dense packing of photosystem I (PSI), photosystem II (PSII), and cytochrome (Cyt) b6f within thylakoid membranes at the molecular level. Furthermore, we functionally tagged PSI, PSII, Cyt b6f, and ATP synthase individually with fluorescent proteins, and revealed the heterogeneous distribution of these four photosynthetic complexes and determined their dynamic features within the crowding membrane environment using live-cell fluorescence imaging. We characterized red light-induced clustering localization and adjustable diffusion of photosynthetic complexes in thylakoid membranes, representative of the reorganization of photosynthetic apparatus in response to environmental changes. Understanding the organization and dynamics of photosynthetic membranes is essential for rational design and construction of artificial photosynthetic systems to underpin bioenergy development. Knowledge of cyanobacterial thylakoid membranes could also be extended to other cell membranes, such as chloroplast and mitochondrial membranes.

Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store-operated Ca2+ entry (SOCE) channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1-mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, a specific Zn2+ chelator, whereas nontumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn2+ exerted rapid inhibitory effects on Orai1-mediated SOCE and on intracellular Ca2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1-mediated intracellular Ca2+ oscillations and reveal a possible molecular basis for zinc-induced cancer prevention and Orai1-SOCE signaling pathway in cancer cells.-Choi, S., Cui, C., Luo, Y., Kim, S.-H., Ko, J.-K., Huo, X., Ma, J., Fu, L.-W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Evaluating Primary Blast Effects In Vitro.

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such blast-induced injuries is of great importance considering the recent trend towards the use of explosives in modern warfare and terrorist-related incidents. To fully understand blast-induced injury, we must first be able to replicate such blast events in a controlled environment using a reproducible method. In this technique using shock tube equipment, shock waves at a range of pressures can be propagated over live cells grown in 2D, and markers of cell viability can be immediately analyzed using a redox indicator assay and the fluorescent imaging of live and dead cells. This method demonstrated that increasing the peak blast overpressure to 127 kPa can stimulate a significant drop in cell viability when compared to untreated controls. Test samples are not limited to adherent cells, but can include cell suspensions, whole-body and tissue samples, through minor modifications to the shock tube setup. Replicating the exact conditions that tissues and cells experience when exposed to a genuine blast event is difficult. Techniques such as the one presented in this article can help to define damage thresholds and identify the transcriptional and epigenetic changes within cells that arise from shock wave exposure.

Evaluating Primary Blast Effects <em>In Vitro</em>

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such blast-induced injuries is of great importance considering the recent trend towards the use of explosives in modern warfare and terrorist-related incidents. To fully understand blast-induced injury, we must first be able to replicate such blast events in a controlled environment using a reproducible method. In this technique using shock tube equipment, shock waves at a range of pressures can be propagated over live cells grown in 2D, and markers of cell viability can be immediately analyzed using a redox indicator assay and the fluorescent imaging of live and dead cells. This method demonstrated that increasing the peak blast overpressure to 127 kPa can stimulate a significant drop in cell viability when compared to untreated controls. Test samples are not limited to adherent cells, but can include cell suspensions, whole-body and tissue samples, through minor modifications to the shock tube setup. Replicating the exact conditions that tissues and cells experience when exposed to a genuine blast event is difficult. Techniques such as the one presented in this article can help to define damage thresholds and identify the transcriptional and epigenetic changes within cells that arise from shock wave exposure.

DNA mimics of red fluorescent proteins (RFP) based on G-quadruplex-confined synthetic RFP chromophores.

Red fluorescent proteins (RFPs) have emerged as valuable biological markers for biomolecule imaging in living systems. Developing artificial fluorogenic systems that mimic RFPs remains an unmet challenge. Here, we describe the design and synthesis of six new chromophores analogous to the chromophores in RFPs. We demonstrate, for the first time, that encapsulating RFP chromophore analogues in canonical DNA G-quadruplexes (G4) can activate bright fluorescence spanning red and far-red spectral regions (Em = 583−668 nm) that nearly match the entire RFP palette. Theoretical calculations and molecular dynamics simulations reveal that DNA G4 greatly restricts radiationless deactivation of chromophores induced by a twisted intramolecular charge transfer (TICT). These DNA mimics of RFP exhibit attractive photophysical properties comparable or superior to natural RFPs, including high quantum yield, large Stokes shifts, excellent anti-photobleaching properties, and two-photon fluorescence. Moreover, these RFP chromophore analogues are a novel and distinctive type of topology-selective G4 probe specific to parallel G4 conformation. The DNA mimics of RFP have been further exploited for imaging of target proteins. Using cancer-specific cell membrane biomarkers as targets, long-term real-time monitoring in single live cell and two-photon fluorescence imaging in tissue sections have been achieved without the need for genetic coding.

Visualisation of an nsPEF induced calcium wave using the genetically encoded calcium indicator GCaMP in U87 human glioblastoma cells

Cytosolic, synthetic chemical calcium indicators are typically used to visualise the rapid increase in intracellular calcium ion concentration that follows nanosecond pulsed electric field (nsPEF) application. This study looks at the application of genetically encoded calcium indicators (GECIs) to investigate the spatiotemporal nature of nsPEF-induced calcium signals using fluorescent live cell imaging. Calcium responses to 44kV/cm, 10ns pulses were observed in U87-MG cells expressing either a plasma membrane targeted GECI (GCaMP5-G), or one cytosolically expressed (GCaMP6-S), and compared to the response of cells loaded with cytosolic or plasma membrane targeted chemical calcium indicators. Application of 100 pulses, to cells containing plasma membrane targeted indicators, revealed a wave of calcium across the cell initiating at the cathode side. A similar spatial wave was not observed with cytosolic indicators with mobile calcium buffering properties. The speed of the wave was related to pulse application frequency and it was not propagated by calcium induced calcium release.

Abstract 009: Mitochondria-targeting Overexpression of AT 2 Receptors Inhibits Intracellular Angiotensin II-induced Respiratory and Glycolytic Stress Responses in Mouse Proximal Tubule Cells

Angiotensin II (ANG II) plays an important role in mitochondrial dysfunction associated with cardiovascular, hypertensive and kidney diseases, but it is not known whether extracellular or intracellular ANG II mediates this effect via activation of cell surface or mitochondrial receptors. As a proof of concept study, we overexpressed an intracellular cyan fluorescent ANG II fusion protein, mito-ANG II, with or without a GFP-tagged full length AT 2 receptor, mito-AT 2 R, selectively in the mitochondria of mouse proximal tubule (mPCT) cells. The mitochondrial respiratory and glycolytic stress responses were measured using Seahorse XF Cell Mito and XF Glycolysis Stress Test Kits, respectively. Live cell fluorescent imaging confirmed the expression and colocalization of mito-ANG II and mito-AT 2 R with a mitochondrial marker MitoTracker®. Overexpression of mito-ANG II for 48 h significantly increased mitochondrial oxygen consumption rate (OCR) by 30% (Control: 239.4 ± 9.2 vs. mito-ANG II: 310.4 ± 12.6 pmol/min; p <0.01, n=5) and extracellular acidification rate (ECAR) by 33% (Control: 6.3 ± 0.3 vs. mito-ANG II: 8.1 ± 0.5 mpH/min; p <0.01, n=5). The effects of mito-ANG II on OCR and ECAR responses were associated with significant increases in phosphorylated MAP kinase ERK1/2, Na + /K + -ATPase, and mitochondrial redox carries, Complex I (NADH coenzyme Q reductase), Complex II (succinate dehydrogenase), Complex III (cytochrome bc 1 complex) and Complex IV (cytochrome c oxidase) ( p <0.01, n=6). The mito-ANG II-induced OCR and ECAR responses were blocked by the AT 1 blocker losartan (10 μM, p <0.01, n=5), but not by the AT 2 receptor blocker PD123319 (10 μM, n.s. , n=5). However, concurrent overexpression of mito-AT 2 R with mito-ANG II in the mitochondria of mPCT cells significantly attenuated the effects of mito-ANG II on OCR and ECAR responses ( p <0.01, n=6), while the effects of mito-AT 2 R overexpression were completely blocked by PD123319 ( p <0.01, n=5). Taken together, our results provide strong evidence that activation of mitochondrial AT 1 receptors by intracellular ANG II stimulates, whereas activation of mitochondrial AT 2 receptors by intracellular ANG II inhibits, mitochondrial respiratory and glycolytic responses in mouse proximal tubule cells.

Infrared neural stimulation induces intracellular Ca2+ release mediated by phospholipase C.

The influence of infrared laser pulses on intracellular Ca2+ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo-4 was used to measure Ca2+ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo-4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca2+ transient with similar dose-response characteristics in hippocampal neurons and glioblastoma cells (half-maximal effective energy density EC50 of around 58 J.cm-2 ). For both type of cells, the source of the infrared-induced Ca2+ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP3 -induced Ca2+ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP3 cell signaling cascade has many potential applications including the development of optoceutical therapeutics.

Tethered Magnets Are the Key to Magnetotaxis: Direct Observations of Magnetospirillum magneticum AMB-1 Show that MamK Distributes Magnetosome Organelles Equally to Daughter Cells

ABSTRACTMagnetotactic bacteria are a unique group of bacteria that synthesize a magnetic organelle termed the magnetosome, which they use to assist with their magnetic navigation in a specific type of bacterial motility called magneto-aerotaxis. Cytoskeletal filaments consisting of the actin-like protein MamK are associated with the magnetosome chain. Previously, the function of MamK was thought to be in positioning magnetosome organelles; this was proposed based on observations via electron microscopy still images. Here, we conducted live-cell time-lapse fluorescence imaging analyses employing highly inclined and laminated optical sheet microscopy, and these methods enabled us to visualize detailed dynamic movement of magnetosomes in growing cells during the entire cell cycle with high-temporal resolution and a high signal/noise ratio. We found that the MamK cytoskeleton anchors magnetosomes through a mechanism that requires MamK-ATPase activity throughout the cell cycle to prevent simple diffusion of magnetosomes within the cell. We concluded that the static chain-like arrangement of the magnetosomes is required to precisely and consistently segregate the magnetosomes to daughter cells. Thus, the daughter cells inherit a functional magnetic sensor that mediates magneto-reception.

Early cytoplasmic uncoating is associated with infectivity of HIV-1

After fusion, HIV delivers its conical capsid into the cytoplasm. To release the contained reverse-transcribing viral genome, the capsid must disassemble in a process termed uncoating. Defining the kinetics, dynamics, and cellular location of uncoating of virions leading to infection has been confounded by defective, noninfectious particles and the stochastic minefield blocking access to host DNA. We used live-cell fluorescent imaging of intravirion fluid phase markers to monitor HIV-1 uncoating at the individual particle level. We find that HIV-1 uncoating of particles leading to infection is a cytoplasmic process that occurs ∼30 min postfusion. Most, but not all, of the capsid protein is rapidly shed in tissue culture and primary target cells, independent of entry pathway. Extended time-lapse imaging with less than one virion per cell allows identification of infected cells by Gag-GFP expression and directly links individual particle behavior to infectivity, providing unprecedented insights into the biology of HIV infection.

Live-cell calcium imaging of adherent and non-adherent GL261 cells reveals phenotype-dependent differences in drug responses

BackgroundThe tumor-derived GL261 cell line is used as a model for studying glioblastoma and other high-grade gliomas, and can be cultured adherently or as free-floating aggregates known as neurospheres. These different culture conditions give rise to distinct phenotypes, with increased tumorigenicity displayed by neurosphere-cultured cells. An important technique for understanding GL261 pathobiology is live cell fluorescent imaging of intracellular calcium. However, live cell imaging of GL261 neurospheres presents a technical challenge, as experimental manipulations where drugs are added to the extracellular media cause the cells to move during analysis. Here we present a method to immobilize GL261 neurospheres with low melting point agarose for calcium imaging using the fluorescent calcium sensor fura-2.MethodsGL261 cells were obtained from the NCI-Frederick Cancer Research Tumor Repository and cultured as adherent cells or induced to form neurospheres by placing freshly trypsinized cells into serum-free media containing fibroblast growth factor 2, epidermal growth factor, and B-27 supplement. Prior to experiments, adherent cells were loaded with fura-2 and cultured on 8-well chamber slides. Non-adherent neurospheres were first loaded with fura-2, placed in droplets onto an 8-well chamber slide, and finally covered with a thin layer of low melting point agarose to immobilize the cells. Ratiometric pseudocolored images were obtained during treatment with ATP, capsaicin, or vehicle control. Cells were marked as responsive if fluorescence levels increased more than 30% above baseline. Differences between treatment groups were tested using Student’s t-tests and one-way ANOVA.ResultsWe found that cellular responses to pharmacological treatments differ based on cellular phenotype. Adherent cells and neurospheres both responded to ATP with a rise in intracellular calcium. Notably, capsaicin treatment led to robust responses in GL261 neurospheres but not adherent cells.ConclusionsWe demonstrate the use of low melting point agarose for immobilizing GL261 cells, a method that is broadly applicable to any cell type cultured in suspension, including acutely trypsinized cells and primary tumor cells. Our results indicate that it is important to consider GL261 phenotype (adherent or neurosphere) when interpreting data regarding physiological responses to experimental compounds.

IDH3 mediates apoptosis of alveolar epithelial cells type 2 due to mitochondrial Ca2+ uptake during hypocapnia

In adult respiratory distress syndrome (ARDS) pulmonary perfusion failure increases physiologic dead-space (VD/VT) correlating with mortality. High VD/VT results in alveolar hypocapnia, which has been demonstrated to cause edema formation, atelectasis, and surfactant depletion, evoked, at least in part, by apoptosis of alveolar epithelial cells (AEC). However, the mechanism underlying the hypocapnia-induced AEC apoptosis is unknown. Here, using fluorescent live-cell imaging of cultured AEC type 2 we could show that in terms of CO2 sensing the tricarboxylic acid cycle enzyme isocitrate dehydrogenase (IDH) 3 seems to be an important player because hypocapnia resulted independently from pH in an elevation of IDH3 activity and subsequently in an increase of NADH, the substrate of the respiratory chain. As a consequence, the mitochondrial transmembrane potential (ΔΨ) rose causing a Ca2+ shift from cytosol into mitochondria, whereas the IDH3 knockdown inhibited these responses. Furthermore, the hypocapnia-induced mitochondrial Ca2+ uptake resulted in reactive oxygen species (ROS) production, and both the mitochondrial Ca2+ uptake and ROS production induced apoptosis. Accordingly, we provide evidence that in AEC type 2 hypocapnia induces elevation of IDH3 activity leading to apoptosis. This finding might give new insight into the pathogenesis of ARDS and may help to develop novel strategies to reduce tissue injury in ARDS.

Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging.

Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences. Fmoc-Trp(C2-BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C-C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH (3-4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6-7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging).

Live-cell imaging of cell signaling using genetically encoded fluorescent reporters.

Synergistic advances in fluorescent protein engineering and live-cell imaging techniques in recent years have fueled the concurrent development and application of genetically encoded fluorescent reporters that are tailored for tracking signaling dynamics in living systems over multiple length and time scales. These biosensors are uniquely suited for this challenging task, owing to their specificity, sensitivity, and versatility, as well as to the noninvasive and nondestructive nature of fluorescence and the power of genetic encoding. Over the past 10 years, a growing number of fluorescent reporters have been developed for tracking a wide range of biological signals in living cells and animals, including second messenger and metabolite dynamics, enzyme activation and activity, and cell cycle progression and neuronal activity. Many of these biosensors are gaining wide use and are proving to be indispensable for unraveling the complex biological functions of individual signaling molecules in their native environment, the living cell, shedding new light on the structural and molecular underpinnings of cell signaling. In this review, we highlight recent advances in protein engineering that are likely to help expand and improve the design and application of these valuable tools. We then turn our focus to specific examples of live-cell imaging using genetically encoded fluorescent reporters as an important platform for advancing our understanding of G protein-coupled receptor signaling and neuronal activity.

Rationally Designed Solvatochromic Fluorescent Indoline Derivatives for Probing Mitochondrial Environment.

A new class of solvatochromic, robust, and multifunctional fluorescent probes derived from indoline is presented. Specificity of mitochondria targeting was achieved and utilized for probing polarity under normal and apoptotic conditions. A large Stokes shift, high quantum yield, thermal, photochemical, and pH stability, tolerance to buffer compositions, and a bioconjugation tool-kit make it a promising candidate for live-cell fluorescence imaging.