Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics.

Real-time live cell imaging and quantification of biothiol dynamics are important for understanding pathophysiological processes. However, the design and synthesis of rational probes that have reversible and real-time capabilities is still challenging. In this work, we have prepared boron-dipyrrolemethene (BODIPY) based fluorescent molecules as ratiometric probes that allow the real-time biothiol dynamics to be observed in living cells. The Michael reaction between α-formyl-BODIPY (BOD-JQ) and GSH exhibited a reversible fluorogenic mechanism with fluorescent emission shifting from 592 nm to 544 nm with t1/2 = 16 ms. In particular, we showed that the probes with targeting agents are capable of detecting biothiols in mitochondria and the endoplasmic reticulum (ER) with high temporal resolution.

Fluorescence imaging is a powerful technique for visualizing biological events in living samples with high temporal and spatial resolution. Fluorescent probes emitting far-red to near infrared (NIR) fluorescence are particularly advantageous for in vivo imaging due to their high tissue permeability and low autofluorescence, as well as their suitability for multicolor imaging. Among the far-red to NIR fluorophores, Si-rhodamine is one of the most practical fluorophores for the development of tailor-made NIR fluorescent probes because of the relative ease of synthesis of various derivatives, the unique intramolecular spirocyclization behavior, and the relatively high water solubility and high photostability of the probes. This review summarizes these features of Si-rhodamines and presents recent advances in the synthesis and applications of far-red to NIR fluorescent probes based on Si-rhodamines, focusing on live-cell imaging applications such as fluorogenic probes, super-resolution imaging and dye-protein hybrid-based indicators.

The ideal fluorescent probe for live-cell imaging is bright and non-cytotoxic and can be delivered easily into the living cells in an efficient manner. The design of synthetic fluorophores having all three of these properties, however, has proved to be challenging. Here, we introduce a simple, yet effective, strategy based on well-established chemistry for designing a new class of fluorescent probes for live-cell imaging. A box-like hybrid cyclophane, namely ExTzBox·4X (6·4X, X = PF6-, Cl-), has been synthesized by connecting an extended viologen (ExBIPY) and a dipyridyl thiazolothiazole (TzBIPY) unit in an end-to-end fashion with two p-xylylene linkers. Photophysical studies show that 6·4Cl has a quantum yield ΦF = 1.00. Furthermore, unlike its ExBIPY2+ and TzBIPY2+ building units, 6·4Cl is non-cytotoxic to RAW 264.7 macrophages, even with a loading concentration as high as 100 μM, presumably on account of its rigid box-like structure which prevents its intercalation into DNA and may inhibit other interactions with it. After gaining an understanding of the toxicity profile of 6·4Cl, we employed it in live-cell imaging. Confocal microscopy has demonstrated that 64+ is taken up by the RAW 264.7 macrophages, allowing the cells to glow brightly with blue laser excitation, without any hint of photobleaching or disruption of normal cell behavior under the imaging conditions. By contrast, the acyclic reference compound Me2TzBIPY·2Cl (4·2Cl) shows very little fluorescence inside the cells, which is quenched completely under the same imaging conditions. In vitro cell investigations underscore the significance of using highly fluorescent box-like rigid cyclophanes for live-cell imaging.

Mitochondrial membrane potential (ΔΨm) is a key indicator of cell health or injury due to its vital roles in adenosine 5'-triphosphate synthesis. Thus, monitoring ΔΨm is of great significance for the assessment of cell status, diagnosis of diseases, and medicament screening. Cationic fluorescent probes suffer from severe photobleaching or false positive signals due to the luminescence of the probe on non-mitochondria. Herein, we report a lipophilic cationic fluorescent probe [1-methyl-2-(4-(1,2,2-triphenylvinyl)styryl)-β-naphthothiazol-1-ium trifluoromethanesulfonate (TPE-NT)] with the features of aggregation-induced emission and intramolecular charge transfer for imaging ΔΨm in live cells. TPE-NT is enriched on the surface of the mitochondrial inner membrane due to the negative ΔΨm, and its fluorescence is activated in the high-viscosity microenvironment. The false positive signals of emission from TPE-NT on non-mitochondria are therefore effectively eliminated. Moreover, TPE-NT exhibits a Stokes shift of >200 nm, near-infrared (∼675 nm) emission, excellent photostability, and low cytotoxicity, which facilitate real-time imaging in live cells. Cell imaging confirmed that the probe can rapidly and reliably report mitochondrial depolarization (decrement of ΔΨm) during cell damage caused by CCCP and H2O2 as well as mitochondrial polarization (increment of ΔΨm) by oligomycin. Furthermore, the probe successfully detected the reduction of ΔΨm in these cell models of hypoxia, heat damage, acidification, aging, inflammation, mitophagy, and apoptosis caused by hypoxia, heatstroke, lactate/pyruvate, doxorubicin, lipopolysaccharide, rapamycin, monensin, and nystatin, respectively.

We carried out live-cell real-time fluorescence imaging to follow the effects of genetic (siRNA) knockdown (KD) of endothelial nitric oxide synthase (eNOS) on mitochondrial biogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). We report here that eNOS KD in hMSCs blocks mitochondrial biogenesis and adipogenesis. The transfer of mitochondria from normal hMSCs to eNOS-deficient hMSCs restores adipogenesis. Furthermore, cell-free mitochondria purified from normal hMSCs also restores adipogenesis in eNOS-deficient cells. Thus, eNOS and NO signaling are essential for mitochondrial biogenesis, and mitochondrial activity is indispensable for adipogenesis in hMSC differentiation. We mapped the path and identified the mechanisms of mitochondrial transfer. We captured real-time images of differentiated mature adipocytes in mitosis and replication. These results reveal that human stem cell-differentiated fat cells are capable of replication. This new finding offers novel insights into our understanding of fat cell expansion and the development of obesity. Real-time imaging in live cells allows synchronized investigation of mitochondrial biogenesis and adipogenesis in stem cell differentiation without reducing living cells to nonliving samples for functional analysis. Live-cell real-time imaging can thus be a faithful and immediate tool for molecular diagnostic medicine. Furthermore, our results suggest that mitochondrial remodeling can be a useful approach in treating adiposity, diabetes, and abnormalities in energy metabolism and vascular signaling.

Oxidative stress resulting from mitochondrial dysfunction may play a role in toxicity caused by many different environmental contaminants, including aromatic hydrocarbons and metal ions, but it has been difficult to evaluate the role of mitochondria in oxidative stress using currently available methods. A new study combining new and established imaging techniques to document mitochondrial dysfunction now indicates this integrated approach to real-time imaging in live cells can be useful for studying the role of oxidative stress in toxicologic responses [EHP 118(7):902–908; Cheng et al.]. Real-time imaging in live cells to detect products of oxidative stress known as reactive oxygen species (ROS) offers superior temporal and spatial resolution compared with traditional methods such as detecting oxidized lipids, proteins, or DNA. But the accuracy and utility of the fluorescent indicator H2DCF-DA, a reagent commonly used for directly detecting ROS in living cells, is limited. The authors conducted a set of experiments in which they exposed cultured human skin carcinoma cells to zinc (Zn2+), a ubiquitous contaminant known to induce oxidative stress. Three different fluorescent imaging techniques were used to study effects of Zn2+ on mitochondria. The first used the fluorophore PG1 to measure production of the ROS hydrogen peroxide. The scientists found that hydrogen peroxide increased within the cells upon Zn2+ exposure and that its production was inhibited with the addition of the mitochondrial inhibitor CCCP, implicating mitochondria as the source of the Zn2+-induced hydrogen peroxide. A second experiment used the fluorescent indicator JC-1 to measure changes in mitochondrial membrane potential (the difference in electrical potential between the inside and outside of the mitochondrial membrane) following exposure to Zn2+. When Zn2+ was administered, loss of JC-1 fluorescence emission indicated a loss of membrane potential consistent with impaired mitochondrial function. A third experiment used the genetically encoded fluorescent sensor MTroGFP1 to measure the redox potential of mitochondria after Zn2+ exposure. MTroGFP1 associates with mitochondria in transfected cells, causing the mitochondria to fluoresce. However, the investigators observed a change in the fluorescent signals following exposure to Zn2+ consistent with a loss of redox potential. In a fourth experiment on mitochondria isolated from live mouse hearts, the authors demonstrated that administering Zn2+ resulted in rapid mitochondrial swelling, another indication of a loss of mitochondrial function. The study shows the value of combining multiple imaging techniques to constitute an integrated approach that permits real-time monitoring of the mechanisms behind oxidative stress within living cells. The results also add to the evidence that oxidative stress induced by Zn2+ originates in mitochondria and sheds light on some of the mechanisms that may be involved. Further study is under way to determine the exact sequence of cellular events by which toxicants induce generation of ROS and mitochondrial dysfunction.

Silafluorene as a promising core for cell-permeant, highly bright and two-photon excitable fluorescent probes for live-cell imaging

Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.

We have developed three types of lipid droplet (LD)-specific fluorescent probes for live-cell imaging, Lipi-Blue, Lipi-Green, and Lipi-Red, which exhibit fluorescence upon being incorporated into LDs both of living and of fixed cells. These Lipi-probes are LD-specific probes that contain a pyrene or perylene group as a fluorescent scaffold and can be used to observe dynamics of LD in live cells and also interrelations with other organelles by simultaneous staining with multiple organelle-specific probes. Additionally, Lipi-Blue and Lipi-Green allow monitoring LDs in live cells even for 48 h after the staining. Here we show that newly formed LDs and previously existed LDs can be separately monitored in a single cell by using these probes and that intercellular transfer of whole LDs is observed in KB cells, but not in HepG2 cells under the same culturing condition. These findings indicate that newly developed LD-specific probes are useful to analyze the dynamics of LDs in live cells.

Chloride plays a crucial role in various cellular functions, and its level is regulated by a variety of chloride transporters and channels. However, to date, we still lack the capability to image instantaneous ion flux through chloride channels at single-cell level. Here, we developed a series of cell-permeable, pH-independent, chloride-sensitive fluorophores for real-time cytosolic chloride imaging, which we call CytoCl dyes. We demonstrated the ability of CytoCl dyes to monitor cytosolic chloride and used it to uncover the rapid changes and transient events of halide flux, which cannot be captured by steady-state imaging. Finally, we successfully imaged the proton-activated chloride channel-mediated ion flux at single-cell level, which is, to our knowledge, the first real-time imaging of ion flux through a chloride channel in unmodified cells. By enabling the imaging of single-cell level ion influx through chloride channels and transporters, CytoCl dyes can expand our understanding of ion flux dynamics, which is critical for characterization and modulator screening of these membrane proteins. A conjugable version of CytoCl dyes was also developed for its customization across different applications.

Live-cell imaging of intracellular proteins enables real-time observation of protein dynamics under near-physiological conditions, providing pivotal insights for both fundamental life science research and medical applications. However, due to limitations such as poor probe permeability and cytotoxicity associated with conventional antibody-based or genetically encoded labeling techniques, live-cell imaging remains a significant challenging. To address these limitations, here in this study, we developed and rigorously validated a novel aptamer-based fluorescent probe for real-time imaging of NEK9 kinase in living cells. First, through in vitro capture-SELEX, a DNA aptamer termed as Apt-011 which could selectively bind NEK9 was identified. Further, capitalizing on the small size, low immunogenicity, and synthetic flexibility of aptamers, we engineered a "signal-on" NEK9-specific aptamer-based fluorescent probe platform. This design leverages the aptamer’s target-­induced conformational change to physically separate the fluorophore-quencher pair, and it has been validated that this fluorescent probe platform could successfully visualize intracellular NEK9 in live-cells without obvious cytotoxicity (cell viability > 95% at working concentrations), offering new opportunities to study NEK9-associated signaling pathways. This work not only provides a robust tool for kinase research but also establishes a generalizable strategy to overcome key bottlenecks in live-cell imaging through rational aptamer engineering.

“Seeing is believing” the proverb well suits for fluorescent imaging probes. Since we can selectively and sensitively visualize small biomolecules, organelles such as lysosomes, neutral molecules, metal ions, anions through cellular imaging, fluorescent probes can help shed light on the physiological and pathophysiological path ways. Since these biomolecules are produced in low concentrations in the biochemical pathways, general analytical techniques either fail to detect or are not sensitive enough to differentiate the relative concentrations. During my Ph.D. study, I exploited synthetic organic techniques to design and synthesize fluorescent probes with desirable properties such as high water solubility, high sensitivity and with varying fluorescent quantum yields. I synthesized a highly water soluble BOIDPY-based turn-on fluorescent probe for endogenous nitric oxide. I also synthesized a series of cell membrane permeable near infrared (NIR) pH activatable fluorescent probes for lysosomal pH sensing. Fluorescent dyes are molecular tools for designing fluorescent bio imaging probes. This prompted me to design and synthesize a hybrid fluorescent dye with a functionalizable chlorine atom and tested the chlorine re-activity for fluorescent probe design. Carbohydrate and protein interactions are key for many biological processes, such as viral and bacterial infections, cell recognition and adhesion, and immune response. Among several analytical techniques aimed to study these interactions, electrochemical bio sensing is more efficient due to its low cost, ease of operation, and possibility for miniaturization. During my Ph.D., I synthesized mannose bearing aniline molecule which is successfully tested as electrochemical bio sensor. A Ferrocene-mannose conjugate with an anchoring group is synthesized, which can be used as a potential electrochemical biosensor.

Imaging the effects of drug delivery in live cells is most often performed by confocal microscopy, which has a resolution limited by the diffraction of light. Here, we report the development of a novel method for imaging drug delivery to live cells using real-time super-resolution microscopy in wide-field mode. Structured illumination microscopy (SIM) is one of several super-resolving microscopy techniques and is capable of imaging beyond the resolution of conventional microscopy to less than 100 nm. In SIM, each super-resolution (SR) image is computationally constructed from a dataset of numerous raw images. Due to the computational time required for SR image construction, datasets are usually post-processed to avoid slowdowns during the acquisition process. Although post-processing is convenient, this approach has limited use since SR images are not available during image acquisition and thus cannot be utilized for real-time evaluation of the sample. Utilizing SR images in real-time is particularly important when objects of interest are smaller than the resolution limit of conventional microscopy. Applications such as determining optimal focus and finding desirable regions of interest are depending on real-time feedback to user. When performing real-time SIM imaging live cells, image acquisition, processing, and the display of SR images must occur in rapid succession thereby reducing the effects of molecular movement. To achieve the data processing speed necessary for real-time imaging of live cells, we utilized a graphics processing unit (GPU) to perform high-speed SR image construction. Each function for processing SIM data was optimized for parallel operation using GPU kernels. Excluding data transfer overhead, the actual GPU processing time for synthesizing the SR image was 45 milliseconds. Compared with parallel processing on a multi-core computer (CPU), using the GPU achieved a 45-fold increase in processing speed. Overall, data throughput using the GPU attained an 8-fold speed increase over CPU processing, thus allowing real-time high resolution imaging. We applied this SIM platform to record the action of nocodazole on microtubule depolymerization in HeLa cells. We are currently developing this real-time imaging method to investigate the delivery and release mechanism of therapeutic cargoes contained within nanoparticles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3982.

The 1,3-dipolar cycloaddition reaction between an alkene and a tetrazole represents one elegant and rare example of fluorophore-forming bioorthogonal chemistry. This is an attractive reaction for imaging applications in live cells that requires less intensive washing steps and/or needs spatiotemporal resolutions. In the present work, as an effort to improve the fluorogenic property of the alkene-tetrazole reaction, an aromatic alkene (styrene) was investigated as the dipolarophile. Over 30-fold improvement in quantum yield of the reaction product was achieved in aqueous solution. According to our mechanistic studies, the observed improvement is likely due to an insufficient protonation of the styrene-tetrazole reaction product. This finding provides useful guidance to the future design of alkene-tetrazole reactions for biological studies. Fluorogenic protein labeling using the styrene-tetrazole reaction was demonstrated both in vitro and in vivo. This was realized by the genetic incorporation of an unnatural amino acid containing the styrene moiety. It is anticipated that the combination of styrene with different tetrazole derivatives can generally improve and broaden the application of alkene-tetrazole chemistry in real-time imaging in live cells.

Nanomaterial-based fluorescent probes for live-cell imaging